Substituted-3H-imidazo [4,5-c] pyridine and 1H-pyrrolo[2,3-c]pyridine series of novel ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) and stimulator for interferon genes (STING) modulators as cancer immunotherapeutics

ABSTRACT

Substituted-3H-imidazo[4,5-c]pyridine and 1H-pyrrolo[2,3-c]pyridine series of novel Ectonucleotide Pyrophosphatase/Phosphodiesterase-1 (ENPP1) and related compounds, which are useful as inhibitors of ENPP1; synthetic methods for making the compounds; pharmaceutical compositions comprising the compounds; and methods of using the compounds and compositions to treat disorders associated with dysfunction of the ENPP1.

BACKGROUND

Ectonucleotide Pyrophophatase/Phosphodiesterase (ENPP) family membersinclude seven isoforms, ENPP1-7, which are type II transmembraneglycoproteins or ectoenzymes. Mass spectrometry and proteomics analysisfrom more than 370 protein targets led to the identification of anextracellular protein ENPP1 as one of the top hit which exhibited highhydrolytic activity. ATP is an identified substrate of ENPP1, which ishydrolyzed to AMP and PPi. CD73 converts AMP to adenosine and inorganicphosphate (Pi). The kinetic experimental data indicates that the ENPP1is capable of hydrolyzing ATP. These ectonucleotide enzymes are involvedin the hydrolysis of pyrophosphate (PPi) and phosphodiester bonds inextracellular nucleotides; such as triphosphates, oligonucleotides andthat generates nucleoside 5′-monophosphates. One of the key isoforms,ENPP1 (Plasma cell membrane glycoprotein-1, PC-1), is involved in anumber of physiological processes, such as development, formation andtrafficking, as well as in pathophysiological conditions. Aberrant ENPP1expression has been detected in breast cancers relative to normalmammary epithelium, an evidence of its potential in the development ofbone metastasis (occurs in approximately 80% cases), Hodgkin's lymphoma,hepatocellular carcinoma, follicular lymphoma, glioblastoma and in othermalignant tumor tissues.

Recent reports suggest that the cyclic dinucleotides (CDNs), a substratefor ENPP1, stimulate innate immunity via STING-dependent activation ofinterferon genes. ENPP1 inhibition of STING pathway activation iscritical for tumor control, similar to that of checkpoint inhibitorssuch as anti PD-1 or PD-L1 which are promising immunotherapeutics forvarious cancers. In addition, mutations in ENPP1 were associated withseveral disorders including infantile arterial calcification(generalized arterial calcification of infancy or GACI), ossification ofthe posterior longitudinal ligament of the spine and insulin signalingand resistance. ENPP1 expression is high in bone and cartilage and isimplicated in lung and kidney fibrosis. A correlation was also foundbetween expression of ENPP1 and the grade of astrocytic tumor. Anotherstudy reported that ENPP1 was required to maintain the undifferentiatedand proliferative state of glioblastoma stem-like cells. Therefore,ENPP1 is an attractive druggable target for the development of novelanticancer, cardiovascular, diabetes, obesity and anti-fibrotictherapeutics.

Importance of ENPP1 activity was further investigated from both directbinding assay and in vitro cellular efficacy on MDA-MB231 cells. ThesiRNA-based knock down of ENPP1 significantly reduced its catalyticactivity both in cell specific and in vivo experiments. Theseexperiments demonstrated that the ENPP1 activity was abolished ontreatment with siRNA. This further supports the validity of this targetin certain diseases. It has been shown recently that thebisphosphothionate analog of endogenous cGAMP is resistant to hydrolysisby ENPP1 phosphodiesterase, and particularly the cyclic dinucleotides(CDNs) are more potent at inducing IFN-β secretion in human THP1 cellsby a mechanism of inhibiting the ENPP1 activity and simultaneous STINGactivation responses.

There is ample evidence that ENPP1 expression is prominent in humanprimary breast tumors relative to normal mammary epithelium, withhighest levels observed in breast-bone metastasis. These data not onlysupport a potential role for ENPP1 in breast-bone metastasis, but alsosupport as a potential prognostic marker for breast cancer. Theseresults from target validation experiments clearly support thepharmacological role of ENPP1 for the development of novelimmunotherapeutics for cancers.

Furthermore, ENPP1 activity has also been implicated in diseases causedby bacteria and/or viruses, and therefore modulators of ENPP1 can beused to treat bacterial and/or viral diseases and conditions.

SUMMARY OF THE INVENTION

The invention, in one aspect, relates to compounds of Formula I:

wherein

-   Y is S;-   Z is —H or —N;-   X₁ is —C═O or —NH,-   X₂ is —C═O or —NH, provided that if X₁ is —C═O, then X₂ is —NH; and    if X₁ is —NH, then X₂ is —C═O;-   R₁ is selected from the group consisting of —H, halogen, —OCF₃,    —CF₃, —CN, OCH₃, and —CH₃;-   R₂ is selected from the group consisting of —H, —NH₂, —OH, and —CH₃;-   R₃ is selected from the group consisting of —H, —NH₂, —OH, and —CH₃;-   R₄ is selected from the group consisting of —H, —CH₂—CH₃ and —CH₃;-   R₅ is selected from the group consisting of

and

-   R₆ is selected from the group consisting of —OCH₃, OH, F, Cl, —CH₃,    O—CH₂—CH₃, and OCH₂—CF₃, OCHF₂,    or an isomer or a pharmaceutically acceptable salt thereof.

In one preferred embodiment,

-   Z is —N;-   X₁ is —C═O;-   X₂ is —NH;-   R₁ is selected from the group consisting of halogen and —CN;-   R₂ is selected from the group consisting of —H and —CH₃;-   R₃ is —H;-   R₄ is —H;-   R₅ is

and

-   R₆ is selected from the group consisting of —OCH₃, and OH.

In a further preferred embodiment, R₁ is selected from the groupconsisting of —Cl and —CN.

Examples of the provided compounds include:

The invention also includes hydrates, solvates, polymorphs, isomers,tautomers of the compounds, pharmaceutically acceptable salts of thecompounds and pharmaceutically acceptable salts of the tautomers.

The invention also provides pharmaceutical formulations, medicamentsincluding the compounds, methods of preparing pharmaceuticalsformulations, medicaments, compounds, and methods of treating patientswith the provided pharmaceutical formulations and compounds.

The compounds of the invention were identified by structure-based,computational docking and binding free energies.

Also disclosed are pharmaceutical compositions comprising atherapeutically effective amount of a disclosed compound and apharmaceutically acceptable carrier.

Also disclosed are synthetic methods for making the disclosed compounds.In a further aspect, disclosed are the products of the disclosedsynthetic methods.

Also disclosed are methods for the treatment of a disorder associatedwith an ENPP1 activity dysfunction in a mammal comprising the step ofadministering to the mammal a therapeutically effective amount of adisclosed compound, or a pharmaceutically acceptable salt, tautomer,isomer, hydrate, solvate, or polymorph thereof.

Also disclosed are methods for inhibition of ENPP1 activity in a mammalcomprising the step of administering to the mammal a therapeuticallyeffective amount of least one disclosed compound, or a pharmaceuticallyacceptable salt, tautomer, isomer, hydrate, solvate, or polymorphthereof.

Also disclosed are methods for inhibiting ENPP1 activity in at least onecell, comprising the step of contacting the at least one cell with aneffective amount of least one disclosed compound, or a pharmaceuticallyacceptable salt, tautomer, isomer, hydrate, solvate, or polymorphthereof.

Also disclosed are methods for treating a disorder associated with anENPP1 activity dysfunction in a mammal through eliciting animmunotherapeutic response in the mammal, comprising administering tothe mammal a therapeutically effective amount of a disclosed compound,or a pharmaceutically acceptable salt, tautomer, isomer, hydrate,solvate, or polymorph thereof, wherein this compound causes animmunotherapeutic response beneficial in the treatment of the disorderassociated with an ENPP1 activity. Such disorder can be, but is notlimited to, any type of cancer or any disease caused by bacteria and/orviruses wherein ENPP1 activity has been implicated.

Also disclosed are pharmaceutical compositions comprising apharmaceutically acceptable carrier and an effective amount of adisclosed compound, or a pharmaceutically acceptable salt, tautomer,isomer, hydrate, solvate, or polymorph thereof.

Also disclosed are kits comprising at least one disclosed compound, or apharmaceutically acceptable salt, tautomer, isomer, hydrate, solvate, orpolymorph thereof.

Also disclosed are methods for manufacturing a medicament comprising,combining at least one disclosed compound or at least one disclosedproduct with a pharmaceutically acceptable carrier or diluent. In afurther aspect, the invention relates to the use of a disclosed compoundin the manufacture of a medicament for the treatment of a a disorderassociated with an ENPP1 activity dysfunction. In a further aspect, theinvention relates to the uses of disclosed compounds in the manufactureof a medicament for the treatment of a a disorder of uncontrolledcellular proliferation.

Also disclosed are uses of a disclosed compound or a disclosed productin the manufacture of a medicament for the treatment of a disorderassociated with an ENPP1 dysfunction in a mammal.

While aspects of the present invention can be described and claimed in aparticular statutory class, such as the system statutory class, this isfor convenience only and one of skill in the art will understand thateach aspect of the present invention can be described and claimed in anystatutory class. Unless otherwise expressly stated, it is in no wayintended that any method or aspect set forth herein be construed asrequiring that its steps be performed in a specific order. Accordingly,where a method claim does not specifically state in the claims ordescriptions that the steps are to be limited to a specific order, it isno way intended that an order be inferred, in any respect. This holdsfor any possible non-express basis for interpretation, including mattersof logic with respect to arrangement of steps or operational flow, plainmeaning derived from grammatical organization or punctuation, or thenumber or type of aspects described in the specification.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a chart of ENPP1 Inhibition Assay for TMP for some of thecompounds of the invention.

FIG. 2A is a chart of a positive control in ENPP2 Inhibition Assay.

FIG. 2B is a chart of ENPP2 Inhibition Assay for some of the compoundsof the invention.

FIG. 3 is a chart of ENPP1 Thermal Shift Assay for a compound of theinvention.

FIG. 4 is a bar chart of ENPP1 Minerazation assay for some of thecompounds of the invention.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

As used herein, nomenclature for compounds, including organic compounds,can be given using common names, IUPAC, IUBMB, or CAS recommendationsfor nomenclature. When one or more stereochemical features are present,Cahn-Ingold-Prelog rules for stereochemistry can be employed todesignate stereochemical priority, E/Z specification, and the like. Oneof skill in the art can readily ascertain the structure of a compound ifgiven a name, either by systemic reduction of the compound structureusing naming conventions, or by commercially available software, such asChemDraw™ (Cambridgesoft Corporation, U.S.A.).

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a functionalgroup,” “an alkyl,” or “a residue” includes mixtures of two or more suchfunctional groups, alkyls, or residues, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, a further aspect includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms a further aspect. It willbe further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that each unit between two particularunits are also disclosed. For example, if 10 and 15 are disclosed, then11, 12, 13, and 14 are also disclosed.

References in the specification and concluding claims to parts by weightof a particular element or component in a composition denotes the weightrelationship between the element or component and any other elements orcomponents in the composition or article for which a part by weight isexpressed. Thus, in a compound containing 2 parts by weight of componentX and 5 parts by weight component Y, X and Y are present at a weightratio of 2:5, and are present in such ratio regardless of whetheradditional components are contained in the compound.

A weight percent (wt. %) of a component, unless specifically stated tothe contrary, is based on the total weight of the formulation orcomposition in which the component is included.

As used herein, the term “ENPP1” refers to EctonucleotidePyrophophatase/Phosphodiesterase.

As used herein, the terms “optional” or “optionally” means that thesubsequently described event or circumstance can or can not occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

As used herein, the term “subject” can be a vertebrate, such as amammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject ofthe herein disclosed methods can be a human, non-human primate, horse,pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The termdoes not denote a particular age or sex. Thus, adult and newbornsubjects, as well as fetuses, whether male or female, are intended to becovered. In one aspect, the subject is a mammal. A patient refers to asubject afflicted with a disease or disorder. The term “patient”includes human and veterinary subjects. In some aspects of the disclosedmethods, the subject has been diagnosed with a need for treatment of adisorder of uncontrolled cellular proliferation associated with an ENPP1dysfunction prior to the administering step. In some aspects of thedisclosed method, the subject has been diagnosed with a need forinhibition of ENPP1 prior to the administering step.

As used herein, the term “treatment” refers to the medical management ofa patient with the intent to cure, ameliorate, stabilize, or prevent adisease, pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder. In various aspects, the term covers anytreatment of a subject, including a mammal (e.g., a human), andincludes: (i) preventing the disease from occurring in a subject thatcan be predisposed to the disease but has not yet been diagnosed ashaving it; (ii) inhibiting the disease, i.e., arresting its development;or (iii) relieving the disease, i.e., causing regression of the disease.In one aspect, the subject is a mammal such as a primate, and, in afurther aspect, the subject is a human. The term “subject” also includesdomesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle,horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse,rabbit, rat, guinea pig, fruit fly, zebra fish etc.).

As used herein, the term “prevent” or “preventing” refers to precluding,averting, obviating, forestalling, stopping, or hindering something fromhappening, especially by advance action. It is understood that wherereduce, inhibit or prevent are used herein, unless specificallyindicated otherwise, the use of the other two words is also expresslydisclosed.

As used herein, the term “diagnosed” means having been subjected to aphysical examination by a person of skill, for example, a physician, andfound to have a condition that can be diagnosed or treated by thecompounds, compositions, or methods disclosed herein. For example,“diagnosed with a disorder of uncontrolled cellular proliferation” meanshaving been subjected to a physical examination by a person of skill,for example, a physician, and found to have a condition that can bediagnosed or treated by a compound or composition that can inhibitENPP1. As a further example, “diagnosed with a need for inhibition ofENPP1” refers to having been subjected to a physical examination by aperson of skill, for example, a physician, and found to have a conditioncharacterized by an ENPP1 dysfunction. Such a diagnosis can be inreference to a disorder, such as a disorder of uncontrolled cellularproliferation, cancer and the like, as discussed herein. For example,“diagnosed with a need for treatment of one or more disorders ofuncontrolled cellular proliferation associated with an ENPP1dysfunction” means having been subjected to a physical examination by aperson of skill, for example, a physician, and found to have one or moredisorders of uncontrolled cellular proliferation associated with anENPP1 dysfunction.

As used herein, the phrase “identified to be in need of treatment for adisorder,” or the like, refers to selection of a subject based upon needfor treatment of the disorder. For example, a subject can be identifiedas having a need for treatment of a disorder (e.g., a disorder relatedto a dysfunction of ENPP1) based upon an earlier diagnosis by a personof skill and thereafter subjected to treatment for the disorder. It iscontemplated that the identification can, in one aspect, be performed bya person different from the person making the diagnosis. It is alsocontemplated, in a further aspect, that the administration can beperformed by one who subsequently performed the administration.

As used herein, the terms “administering” and “administration” refer toany method of providing a pharmaceutical preparation to a subject. Suchmethods are well known to those skilled in the art and include, but arenot limited to, oral administration, transdermal administration,administration by inhalation, nasal administration, topicaladministration, intravaginal administration, ophthalmic administration,intraaural administration, intracerebral administration, rectaladministration, sublingual administration, buccal administration,intraurethral administration, and parenteral administration, includinginjectable such as intravenous administration, intra-arterialadministration, intramuscular administration, and subcutaneousadministration. Administration can be continuous or intermittent. Invarious aspects, a preparation can be administered therapeutically; thatis, administered to treat an existing disease or condition. In furthervarious aspects, a preparation can be administered prophylactically;that is, administered for prevention of a disease or condition.

The term “contacting” as used herein refers to bringing a disclosedcompound and a cell, target receptor, or other biological entitytogether in such a manner that the compound can affect the activity ofthe target (e.g., receptor, cell, etc.), either directly; i.e., byinteracting with the target itself, or indirectly; i.e., by interactingwith another molecule, co-factor, factor, or protein on which theactivity of the target is dependent.

As used herein, the terms “effective amount” and “amount effective”refer to an amount that is sufficient to achieve the desired result orto have an effect on an undesired condition. For example, a“therapeutically effective amount” refers to an amount that issufficient to achieve the desired therapeutic result or to have aneffect on undesired symptoms, but is generally insufficient to causeadverse side affects. The specific therapeutically effective dose levelfor any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the specific composition employed; the age, body weight, general health,sex, and diet of the patient; the time of administration; the route ofadministration; the rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination or coincidentalwith the specific compound employed and like factors well known in themedical arts. For example, it is well within the skill of the art tostart doses of a compound at levels lower than those required to achievethe desired therapeutic effect and to gradually increase the dosageuntil the desired effect is achieved. If desired, the effective dailydose can be divided into multiple doses for purposes of administration.Consequently, single dose compositions can contain such amounts orsubmultiples thereof to make up the daily dose. The dosage can beadjusted by the individual physician in the event of anycontraindications. Dosage can vary, and can be administered in one ormore dose administrations daily, for one or several days. Guidance canbe found in the literature for appropriate dosages for given classes ofpharmaceutical products. In further various aspects, a preparation canbe administered in a “prophylactically effective amount”; that is, anamount effective for prevention of a disease or condition.

As used herein, “EC₅₀,” is intended to refer to the concentration of asubstance (e.g., a compound or a drug) that is required for 50% agonismor activation of a biological process, or component of a process,including a protein, subunit, organelle, ribonucleoprotein, etc. In oneaspect, an EC₅₀ can refer to the concentration of a substance that isrequired for 50% agonism or activation in vivo, as further definedelsewhere herein. In a further aspect, EC₅₀ refers to the concentrationof agonist or activator that provokes a response halfway between thebaseline and maximum response.

As used herein, “IC₅₀,” is intended to refer to the concentration of asubstance (e.g., a compound or a drug) that is required for 50%inhibition of a biological process, or component of a process, includinga protein, subunit, organelle, ribonucleoprotein, etc. For example, anIC₅₀ can refer to the concentration of a substance that is required for50% inhibition in vivo or the inhibition is measured in vitro, asfurther defined elsewhere herein. Alternatively, IC₅₀ refers to the halfmaximal (50%) inhibitory concentration (IC) of a substance. Theinhibition can be measured in a cell-line such as AN3 CA, BT-20, BT-549,HCT 116, HER218, MCF7, MDA-MB-231, MDA-MB-235, MDA-MB-435S, MDA-MB-468,PANG-1, PC-3, SK-N-MC, T-47D, and U-87 MG.

The term “pharmaceutically acceptable” describes a material that is notbiologically or otherwise undesirable, i.e., without causing anunacceptable level of undesirable biological effects or interacting in adeleterious manner.

The term “stable,” as used herein, refers to compounds that are notsubstantially altered when subjected to conditions to allow for theirproduction, detection, and, in certain aspects, their recovery,purification, and use for one or more of the purposes disclosed herein.

As used herein, the term “derivative” refers to a compound having astructure derived from the structure of a parent compound (e.g., acompound disclosed herein) and whose structure is sufficiently similarto those disclosed herein and based upon that similarity, would beexpected by one skilled in the art to exhibit the same or similaractivities and utilities as the claimed compounds, or to induce, as aprecursor, the same or similar activities and utilities as the claimedcompounds. Exemplary derivatives include salts, esters, amides, salts ofesters or amides, and N-oxides of a parent compound.

As used herein, the term “pharmaceutically acceptable carrier” refers tosterile aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, as well as sterile powders for reconstitution into sterileinjectable solutions or dispersions just prior to use. Examples ofsuitable aqueous and nonaqueous carriers, diluents, solvents or vehiclesinclude water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol and the like), carboxymethylcellulose and suitablemixtures thereof, vegetable oils (such as olive oil) and injectableorganic esters such as ethyl oleate. Proper fluidity can be maintained,for example, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions andby the use of surfactants. These compositions can also contain adjuvantssuch as preservatives, wetting agents, emulsifying agents and dispersingagents. Prevention of the action of microorganisms can be ensured by theinclusion of various antibacterial and antifungal agents such asparaben, chlorobutanol, phenol, sorbic acid and the like. It can also bedesirable to include isotonic agents such as sugars, sodium chloride andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the inclusion of agents, such as aluminummonostearate and gelatin, which delay absorption. Injectable depot formsare made by forming microencapsule matrices of the drug in biodegradablepolymers such as polylactide-polyglycolide, poly(orthoesters) andpoly(anhydrides). Depending upon the ratio of drug to polymer and thenature of the particular polymer employed, the rate of drug release canbe controlled. Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues. The injectable formulations can be sterilized, forexample, by filtration through a bacterial-retaining filter or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable media just prior to use. Suitable inertcarriers can include sugars such as lactose. Desirably, at least 95% byweight of the particles of the active ingredient have an effectiveparticle size in the range of 0.01 to 10 micrometers.

A residue of a chemical species, as used in the specification andconcluding claims, refers to the moiety that is the resulting product ofthe chemical species in a particular reaction scheme or subsequentformulation or chemical product, regardless of whether the moiety isactually obtained from the chemical species. Thus, an ethylene glycolresidue in a polyester refers to one or more —OCH₂CH₂O— units in thepolyester, regardless of whether ethylene glycol was used to prepare thepolyester. Similarly, a sebacic acid residue in a polyester refers toone or more —CO(CH₂)₈CO— moieties in the polyester, regardless ofwhether the residue is obtained by reacting sebacic acid or an esterthereof to obtain the polyester.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, and aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described below. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this disclosure, the heteroatoms, such as nitrogen, canhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. This disclosure is not intended to be limited in any mannerby the permissible substituents of organic compounds. Also, the terms“substitution” or “substituted with” include the implicit proviso thatsuch substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., a compound that does not spontaneouslyundergo transformation such as by rearrangement, cyclization,elimination, etc. It is also contemplated that, in certain aspects,unless expressly indicated to the contrary, individual substituents canbe further optionally substituted (i.e., further substituted orunsubstituted).

In defining various terms, “A¹,” “A²,” “A³,” and “A⁴” are used herein asgeneric symbols to represent various specific substituents. Thesesymbols can be any substituent, not limited to those disclosed herein,and when they are defined to be certain substituents in one instance,they can, in another instance, be defined as some other substituents.

The term “alkyl” as used herein is a branched or unbranched saturatedhydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl,isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl,dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. Thealkyl group can be cyclic or acyclic. The alkyl group can be branched orunbranched. The alkyl group can also be substituted or unsubstituted.For example, the alkyl group can be substituted with one or more groupsincluding, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether,halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein.A “lower alkyl” group is an alkyl group containing from one to six(e.g., from one to four) carbon atoms.

For example, a “C1-C3 alkyl” group can be selected from methyl, ethyl,n-propyl, propyl, and cyclopropyl, or from a subset thereof. In certainaspects, the “C1-C3 alkyl” group can be optionally further substituted.As a further example, a “C1-C4 alkyl” group can be selected from methyl,ethyl, n-propyl, i-propyl, cyclopropyl, n-butyl, i-butyl, s-butyl,t-butyl, and cyclobutyl, or from a subset thereof. In certain aspects,the “C1-C4 alkyl” group can be optionally further substituted. As afurther example, a “C1-C6 alkyl” group can be selected from methyl,ethyl, n-propyl, i-propyl, cyclopropyl, n-butyl, i-butyl, s-butyl,t-butyl, cyclobutyl, n-pentyl, i-pentyl, s-pentyl, t-pentyl, neopentyl,cyclopentyl, n-hexyl, i-hexyl, 3-methylpentane, 2,3-dimethylbutane,neohexane, and cyclohexane, or from a subset thereof. In certainaspects, the “C1-C6 alkyl” group can be optionally further substituted.As a further example, a “C1-C8 alkyl” group can be selected from methyl,ethyl, n-propyl, i-propyl, cyclopropyl, n-butyl, i-butyl, s-butyl,t-butyl, cyclobutyl, n-pentyl, i-pentyl, s-pentyl, t-pentyl, neopentyl,cyclopentyl, n-hexyl, i-hexyl, 3-methylpentane, 2,3-dimethylbutane,neohexane, cyclohexane, heptane, cycloheptane, octane, and cyclooctane,or from a subset thereof. In certain aspects, the “C1-C8 alkyl” groupcan be optionally further substituted. As a further example, a “C1-C12alkyl” group can be selected from methyl, ethyl, n-propyl, i-propyl,cyclopropyl, n-butyl, i-butyl, s-butyl, t-butyl, cyclobutyl, n-pentyl,i-pentyl, s-pentyl, t-pentyl, neopentyl, cyclopentyl, n-hexyl, t-hexyl,3-methylpentane, 2,3-dimethylbutane, neohexane, cyclohexane, heptane,cycloheptane, octane, cyclooctane, nonane, cyclononane, decane,cyclodecane, undecane, cycloundecane, dodecane, and cyclododecane, orfrom a subset thereof. In certain aspects, the “C1-C12 alkyl” group canbe optionally further substituted.

Throughout the specification “alkyl” is generally used to refer to bothunsubstituted alkyl groups and substituted alkyl groups; however,substituted alkyl groups are also specifically referred to herein byidentifying the specific substituent(s) on the alkyl group. For example,the term “halogenated alkyl” or “haloalkyl” specifically refers to analkyl group that is substituted with one or more halide, e.g., fluorine,chlorine, bromine, or iodine. The term “alkoxyalkyl” specifically refersto an alkyl group that is substituted with one or more alkoxy groups, asdescribed below. The term “alkylamino” specifically refers to an alkylgroup that is substituted with one or more amino groups, as describedbelow, and the like. When “alkyl” is used in one instance and a specificterm such as “alkylalcohol” is used in another, it is not meant to implythat the term “alkyl” does not also refer to specific terms such as“alkylalcohol” and the like.

This practice is also used for other groups described herein. That is,while a term such as “cycloalkyl” refers to both unsubstituted andsubstituted cycloalkyl moieties, the substituted moieties can, inaddition, be specifically identified herein; for example, a particularsubstituted cycloalkyl can be referred to as, e.g., an“alkylcycloalkyl.” Similarly, a substituted alkoxy can be specificallyreferred to as, e.g., a “halogenated alkoxy,” a particular substitutedalkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, thepractice of using a general term, such as “cycloalkyl,” and a specificterm, such as “alkylcycloalkyl,” is not meant to imply that the generalterm does not also include the specific term.

The term “cycloalkyl” as used herein is a non-aromatic carbon-based ringcomposed of at least three carbon atoms. Examples of cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, norbornyl, and the like. The term “heterocycloalkyl” is atype of cycloalkyl group as defined above, and is included within themeaning of the term “cycloalkyl,” where at least one of the carbon atomsof the ring is replaced with a heteroatom such as, but not limited to,nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group andheterocycloalkyl group can be substituted or unsubstituted. Thecycloalkyl group and heterocycloalkyl group can be substituted with oneor more groups including, but not limited to, alkyl, cycloalkyl, alkoxy,amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, nitrile,sulfonamide, or thiol as described herein.

The term “aryl” as used herein is a group that contains any carbon-basedaromatic group including, but not limited to, benzene, naphthalene,phenyl, biphenyl, phenoxybenzene, and the like. The term “aryl” alsoincludes “heteroaryl,” which is defined as a group that contains anaromatic group that has at least one heteroatom incorporated within thering of the aromatic group. Examples of heteroatoms include, but are notlimited to, nitrogen, oxygen, sulfur, and phosphorus. Likewise, the term“non-heteroaryl,” which is also included in the term “aryl,” defines agroup that contains an aromatic group that does not contain aheteroatom. The aryl group can be substituted or unsubstituted. The arylgroup can be substituted with one or more groups including, but notlimited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, nitrile,sulfonamide, or thiol as described herein. The term “biaryl” is aspecific type of aryl group and is included in the definition of “aryl.”Biaryl refers to two aryl groups that are bound together via a fusedring structure, as in naphthalene, or are attached via one or morecarbon-carbon bonds, as in biphenyl.

The terms “halogen,” “halide,” and “halo,” as used herein, refer to thehalogens fluorine, chlorine, bromine, and iodine. It is alsocontemplated that, in various aspects, halogen can be selected fromfluoro, chloro, bromo, and iodo. For example, halogen can be selectedfrom fluoro, chloro, and bromo. As a further example, halogen can beselected from fluoro and chloro. As a further example, halogen can beselected from chloro and bromo. As a further example, halogen can beselected from bromo and iodo. As a further example, halogen can beselected from chloro, bromo, and iodo. In one aspect, halogen can befluoro. In a further aspect, halogen can be chloro. In a still furtheraspect, halogen is bromo. In a yet further aspect, halogen is iodo.

It is also contemplated that, in certain aspects, pseudohalogens (e.g.triflate, mesylate, tosylate, brosylate, etc.) can be used in place ofhalogens. For example, in certain aspects, halogen can be replaced bypseudohalogen. As a further example, pseudohalogen can be selected fromtriflate, mesylate, tosylate, and brosylate. In one aspect,pseudohalogen is triflate. In a further aspect, pseudohalogen ismesylate. In a further aspect, pseudohalogen is tosylate. In a furtheraspect, pseudohalogen is brosylate.

The term “heterocycle,” as used herein refers to single and multi-cyclicaromatic or non-aromatic ring systems in which at least one of the ringmembers is other than carbon. Heterocycle includes azetidine, dioxane,furan, imidazole, isothiazole, isoxazole, morpholine, oxazole, oxazole,including, 1,2,3-oxadiazole, 1,2,5-oxadiazole and 1,3,4-oxadiazole,piperazine, piperidine, pyrazine, pyrazole, pyridazine, pyridine,pyrimidine, pyrrole, pyrrolidine, tetrahydropyran, tetrahydropyran,tetrazine, including 1,2,4,5-tetrazine, tetrazole, including1,2,3,4-tetrazole and 1,2,4,5-tetrazole, thiadiazole, including,1,2,3-thiadiazole, 1,2,5-thiadiazole, and 1,3,4-thiadiazole, thiazole,thiophene, triazine, including 1,3,5-triazine and 1,2,4-triazine,triazole, including, 1,2,3-triazole, 1,3,4-triazole, and the like.

The term “hydroxyl” as used herein is represented by the formula —OH.

“R¹,” “R²,” “R³,” “R^(n),” where n is an integer, as used herein can,independently, possess one or more of the groups listed above. Forexample, if R¹ is a straight chain alkyl group, one of the hydrogenatoms of the alkyl group can optionally be substituted with a hydroxylgroup, an alkoxy group, an alkyl group, a halide, and the like.Depending upon the groups that are selected, a first group can beincorporated within second group or, alternatively, the first group canbe pendant (i.e., attached) to the second group. For example, with thephrase “an alkyl group comprising an amino group,” the amino group canbe incorporated within the backbone of the alkyl group. Alternatively,the amino group can be attached to the backbone of the alkyl group. Thenature of the group(s) that is (are) selected will determine if thefirst group is embedded or attached to the second group.

As described herein, compounds of the invention may contain “optionallysubstituted” moieties. In general, the term “substituted,” whetherpreceded by the term “optionally” or not, means that one or morehydrogens of the designated moiety are replaced with a suitablesubstituent. Unless otherwise indicated, an “optionally substituted”group may have a suitable substituent at each substitutable position ofthe group, and when more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds. In is also contemplated that, in certain aspects,unless expressly indicated to the contrary, individual substituents canbe further optionally substituted (i.e., further substituted orunsubstituted).

Compounds described herein can contain one or more double bonds and,thus, potentially give rise to cis/trans (E/Z) isomers, as well as otherconformational isomers. Unless stated to the contrary, the inventionincludes all such possible isomers, as well as mixtures of such isomers.

Unless stated to the contrary, a formula with chemical bonds shown onlyas solid lines and not as wedges or dashed lines contemplates eachpossible isomer, e.g., each enantiomer and diastereomer, and a mixtureof isomers, such as a racemic or scalemic mixture. Compounds describedherein can contain one or more asymmetric centers and, thus, potentiallygive rise to diastereomers and optical isomers. Unless stated to thecontrary, the present invention includes all such possible diastereomersas well as their racemic mixtures, their substantially pure resolvedenantiomers, all possible geometric isomers, and pharmaceuticallyacceptable salts thereof. Mixtures of stereoisomers, as well as isolatedspecific stereoisomers, are also included. During the course of thesynthetic procedures used to prepare such compounds, or in usingracemization or epimerization procedures known to those skilled in theart, the products of such procedures can be a mixture of stereoisomers.

Many organic compounds exist in optically active forms having theability to rotate the plane of plane-polarized light. In describing anoptically active compound, the prefixes D and L or R and S are used todenote the absolute configuration of the molecule about its chiralcenter(s). The prefixes d and or (+) and (−) are employed to designatethe sign of rotation of plane-polarized light by the compound, with (−)or l meaning that the compound is levorotatory. A compound prefixed with(+) or d is dextrorotatory. For a given chemical structure, thesecompounds, called stereoisomers, are identical except that they arenon-superimposable mirror images of one another. A specific stereoisomercan also be referred to as an enantiomer, and a mixture of such isomersis often called an enantiomeric mixture. A 50:50 mixture of enantiomersis referred to as a racemic mixture.

Many of the compounds described herein can have one or more chiralcenters and therefore can exist in different enantiomeric forms. Ifdesired, a chiral carbon can be designated with an asterisk (*). Whenbonds to the chiral carbon are depicted as straight lines in thedisclosed formulas, it is understood that both the (R) and (S)configurations of the chiral carbon, and hence both enantiomers andmixtures thereof, are embraced within the formula. As is used in theart, when it is desired to specify the absolute configuration about achiral carbon, one of the bonds to the chiral carbon can be depicted asa wedge (bonds to atoms above the plane) and the other can be depictedas a series or wedge of short parallel lines is (bonds to atoms belowthe plane). The Cahn-Inglod-Prelog system can be used to assign the (R)or (S) configuration to a chiral carbon.

Compounds described herein comprise atoms in both their natural isotopicabundance and in non-natural abundance. The disclosed compounds can beisotopically-labelled or isotopically-substituted compounds identical tothose described, but for the fact that one or more atoms are replaced byan atom having an atomic mass or mass number different from the atomicmass or mass number typically found in nature. Examples of isotopes thatcan be incorporated into compounds of the invention include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine,such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ¹⁸F and ³⁶Cl,respectively. Compounds further comprise prodrugs thereof, andpharmaceutically acceptable salts of said compounds or of said prodrugswhich contain the aforementioned isotopes and/or other isotopes of otheratoms are within the scope of this invention. Certainisotopically-labelled compounds of the present invention, for examplethose into which radioactive isotopes such as ³H and ¹⁴C areincorporated, are useful in drug and/or substrate tissue distributionassays. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C, isotopes areparticularly preferred for their ease of preparation and detectability.Further, substitution with heavier isotopes such as deuterium, i.e., ²H,can afford certain therapeutic advantages resulting from greatermetabolic stability, for example increased in vivo half-life or reduceddosage requirements and, hence, may be preferred in some circumstances.Isotopically labelled compounds of the present invention and prodrugsthereof can generally be prepared by carrying out the procedures below,by substituting a readily available isotopically labelled reagent for anon-isotopically labelled reagent.

The compounds described in the invention can be present as a solvate. Insome cases, the solvent used to prepare the solvate is an aqueoussolution, and the solvate is then often referred to as a hydrate. Thecompounds can be present as a hydrate, which can be obtained, forexample, by crystallization from a solvent or from aqueous solution. Inthis connection, one, two, three or any arbitrary number of solvate orwater molecules can combine with the compounds according to theinvention to form solvates and hydrates. Unless stated to the contrary,the invention includes all such possible solvates.

It is also appreciated that certain compounds described herein can bepresent as an equilibrium of tautomers. For example, ketones with anα-hydrogen can exist in an equilibrium of the keto form and the enolform.

Likewise, amides with an N-hydrogen can exist in an equilibrium of theamide form and the imidic acid form. Unless stated to the contrary, theinvention includes all such possible tautomers.

It is known that chemical substances form solids which are present indifferent states of order which are termed polymorphic forms ormodifications. The different modifications of a polymorphic substancecan differ greatly in their physical properties. The compounds accordingto the invention can be present in different polymorphic forms, with itbeing possible for particular modifications to be metastable. Unlessstated to the contrary, the invention includes all such possiblepolymorphic forms.

In some aspects, a structure of a compound can be represented by aformula:

which is understood to be equivalent to a formula:

wherein n is typically an integer. That is, R^(n) is understood torepresent five independent substituents, R^(n(a)), R^(n(b)), R^(n(c)),R^(n(d)), R^(n(e)). By “independent substituents,” it is meant that eachR substituent can be independently defined. For example, if in oneinstance R^(n(a)) is halogen, then R^(n(b)) is not necessarily halogenin that instance.

Certain materials, compounds, compositions, and components disclosedherein can be obtained commercially or readily synthesized usingtechniques generally known to those of skill in the art. For example,the starting materials and reagents used in preparing the disclosedcompounds and compositions are either available from commercialsuppliers such as Sigma-Aldrich Chemical Co., (Milwaukee, Wis.), AcrosOrganics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), orSigma (St. Louis, Mo.) or are prepared by methods known to those skilledin the art following procedures set forth in references such as Fieserand Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wileyand Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced OrganicChemistry, (John Wiley and Sons, 4th Edition); and Larock'sComprehensive Organic Transformations (VCH Publishers Inc., 1989).

Unless otherwise expressly stated, it is in no way intended that anymethod set forth herein be construed as requiring that its steps beperformed in a specific order. Accordingly, where a method claim doesnot actually recite an order to be followed by its steps or it is nototherwise specifically stated in the claims or descriptions that thesteps are to be limited to a specific order, it is no way intended thatan order be inferred, in any respect. This holds for any possiblenon-express basis for interpretation, including: matters of logic withrespect to arrangement of steps or operational flow; plain meaningderived from grammatical organization or punctuation; and the number ortype of embodiments described in the specification.

Disclosed are the components to be used to prepare the compositions ofthe invention as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds can not be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular compound is disclosed and discussed and anumber of modifications that can be made to a number of moleculesincluding the compounds are discussed, specifically contemplated is eachand every combination and permutation of the compound and themodifications that are possible unless specifically indicated to thecontrary. Thus, if a class of molecules A, B, and C are disclosed aswell as a class of molecules D, E, and F and an example of a combinationmolecule, A-D is disclosed, then even if each is not individuallyrecited each is individually and collectively contemplated meaningcombinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considereddisclosed. Likewise, any subset or combination of these is alsodisclosed. Thus, for example, the sub-group of A-E, B-F, and C-E wouldbe considered disclosed. This concept applies to all aspects of thisapplication including, but not limited to, steps in methods of makingand using the compositions of the invention. Thus, if there are avariety of additional steps that can be performed it is understood thateach of these additional steps can be performed with any specificembodiment or combination of embodiments of the methods of theinvention.

It is understood that the compositions disclosed herein have certainfunctions. Disclosed herein are certain structural requirements forperforming the disclosed functions, and it is understood that there area variety of structures that can perform the same function that arerelated to the disclosed structures, and that these structures willtypically achieve the same result.

B. Compounds

In one aspect, the invention relates to compounds useful as inhibitorsof ENPP1. Moreover, in one aspect, the compounds of the invention areuseful in the treatment of disorders of uncontrolled cellularproliferations. In a further aspect, the disorder of uncontrolledcellular proliferation is a cancer or a tumor. In a still furtheraspect, the disorder of uncontrolled cellular proliferation isassociated with an ENPP1 dysfunction, as further described herein.

In another aspect, the compounds of the invention are useful in thetreatment of diseases of bacterial or viral origin. Accordingly, in oneaspect, the invention provides a method of treating a disease caused bybacteria or viruses, comprising administering to a subject atherapeutically effective amount of a compound of the invention.

It is contemplated that each disclosed derivative can be optionallyfurther substituted. It is also contemplated that any one or morederivative can be optionally omitted from the invention. It isunderstood that a disclosed compound can be provided by the disclosedmethods. It is also understood that the disclosed compounds can beemployed in the disclosed methods of using.

1. Structure

In one aspect, the invention relates to a compound of Formula I:

wherein

-   Y is S;-   Z is —H or —N;-   X₁ is —C═O or —NH,-   X₂ is —C═O or —NH, provided that if X₁ is —C═O, then X₂ is —NH; and    if X₁ is —NH, then X₂ is —C═O;-   R₁ is selected from the group consisting of —H, halogen, —OCF₃,    —CF₃, —CN, OCH₃, and —CH₃;-   R₂ is selected from the group consisting of —H, —NH₂, —OH, and —CH₃;-   R₃ is selected from the group consisting of —H, —NH₂, —OH, and —CH₃;-   R₄ is selected from the group consisting of —H, —CH₂—CH₃ and —CH₃;-   R₅ is selected from the group consisting of

and

-   R₆ is selected from the group consisting of —OCH₃, OH, F, Cl, —CH₃,    O—CH₂—CH₃, and OCH₂—CF₃, OCHF₂,    or an isomer or a pharmaceutically acceptable salt thereof.

In one preferred embodiment,

-   Z is —N;-   X₁ is —C═O;-   X₂ is —NH;-   R₁ is selected from the group consisting of halogen and —CN;-   R₂ is selected from the group consisting of —H and —CH₃;-   R₃ is —H;-   R₄ is —H;-   R₅ is

and

-   R₆ is selected from the group consisting of —OCH₃, and OH.

In a further preferred embodiment, R₁ is selected from the groupconsisting of —Cl and —CN.

Examples of the provided compounds include:

The invention also provides a pharmaceutical composition comprising atherapeutically effective amount of any of a compound of the inventionand a pharmaceutically acceptable carrier.

The invention also provides a method for the treatment of a disorder ofuncontrolled cellular proliferation in a mammal, the method comprisingthe step of administering to the mammal an effective amount of any ofthe compounds of the invention.

The invention also provides a method for decreasing ENPP1 activity in amammal, the method comprising the step of administering to the mammal aneffective amount of any of the compounds of the invention.

The invention also provides a method for inhibiting ENPP1 activity in amammal, the method comprising the step of administering to the mammal aneffective amount of any of the compounds of the invention.

C. Methods of Making the Compounds

In one aspect, the invention relates to methods of making compoundsuseful as inhibitors of ENPP1. In a further aspect, the products ofdisclosed methods of making are modulators of ENPP1 activity.

The compounds of this invention can be prepared by employing reactionsas shown in the following schemes, in addition to other standardmanipulations that are known in the literature, exemplified in theexperimental sections or clear to one skilled in the art. For clarity,examples having a single substituent are shown where multiplesubstituents are allowed under the definitions disclosed herein.

Reactions used to generate the compounds of this invention are preparedby employing reactions as shown in the following Reaction Schemes, inaddition to other standard manipulations known in the literature or toone skilled in the art. The following examples are provided so that theinvention might be more fully understood, are illustrative only, andshould not be construed as limiting.

In one aspect, the disclosed compounds comprise the products of thesynthetic methods described herein. In a further aspect, the disclosedcompounds comprise a compound produced by a synthetic method describedherein. In a still further aspect, the invention comprises apharmaceutical composition comprising a therapeutically effective amountof the product of the disclosed methods and a pharmaceuticallyacceptable carrier. In a still further aspect, the invention comprises amethod for manufacturing a medicament comprising combining at least onecompound of any of disclosed compounds or at least one product of thedisclosed methods with a pharmaceutically acceptable carrier or diluent.

Where reaction conditions and amounts of ingredients are not stated, itis believed that it is within a skill in the art to determine them. Itis contemplated that each disclosed methods can further compriseadditional steps, manipulations, and/or components. It is alsocontemplated that any one or more step, manipulation, and/or componentcan be optionally omitted from the invention. It is understood that adisclosed methods can be used to provide the disclosed compounds. It isalso understood that the products of the disclosed methods can beemployed in the disclosed methods of using.

D. Pharmaceutical Compositions

In one aspect, the invention relates to pharmaceutical compositionscomprising the disclosed compounds. That is, a pharmaceuticalcomposition can be provided comprising a therapeutically effectiveamount of at least one disclosed compound or at least one product of adisclosed method and a pharmaceutically acceptable carrier.

In a further aspect, the invention relates to pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier and aneffective amount of the product of a disclosed synthetic method. In afurther aspect, the effective amount is a therapeutically effectiveamount. In a further aspect, the effective amount is a prophylacticallyeffective amount. In a further aspect, the compound is a disclosedcompound.

In certain aspects, the disclosed pharmaceutical compositions comprisethe disclosed compounds (including pharmaceutically acceptable salt(s)thereof) as an active ingredient, a pharmaceutically acceptable carrier,and, optionally, other therapeutic ingredients or adjuvants. The instantcompositions include those suitable for oral, rectal, topical, andparenteral (including subcutaneous, intramuscular, and intravenous)administration, although the most suitable route in any given case willdepend on the particular host, and nature and severity of the conditionsfor which the active ingredient is being administered. Thepharmaceutical compositions can be conveniently presented in unit dosageform and prepared by any of the methods well known in the art ofpharmacy.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts prepared from pharmaceutically acceptable non-toxic bases oracids. When the compound of the present invention is acidic, itscorresponding salt can be conveniently prepared from pharmaceuticallyacceptable non-toxic bases, including inorganic bases and organic bases.Salts derived from such inorganic bases include aluminum, ammonium,calcium, copper (-ic and -ous), ferric, ferrous, lithium, magnesium,manganese (-ic and -ous), potassium, sodium, zinc and the like salts.Particularly preferred are the ammonium, calcium, magnesium, potassiumand sodium salts. Salts derived from pharmaceutically acceptable organicnon-toxic bases include salts of primary, secondary, and tertiaryamines, as well as cyclic amines and substituted amines such asnaturally occurring and synthesized substituted amines. Otherpharmaceutically acceptable organic non-toxic bases from which salts canbe formed include ion exchange resins such as, for example, arginine,betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like.

As used herein, the term “pharmaceutically acceptable non-toxic acids”,includes inorganic acids, organic acids, and salts prepared therefrom,for example, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric,ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric,isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic,nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric,p-toluenesulfonic acid and the like. Preferred are citric, hydrobromic,hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.

In practice, the compounds of the invention, or pharmaceuticallyacceptable salts thereof, of this invention can be combined as theactive ingredient in intimate admixture with a pharmaceutical carrieraccording to conventional pharmaceutical compounding techniques. Thecarrier can take a wide variety of forms depending on the form ofpreparation desired for administration, e.g., oral or parenteral(including intravenous). Thus, the pharmaceutical compositions of thepresent invention can be presented as discrete units suitable for oraladministration such as capsules, cachets or tablets each containing apredetermined amount of the active ingredient. Further, the compositionscan be presented as a powder, as granules, as a solution, as asuspension in an aqueous liquid, as a non-aqueous liquid, as anoil-in-water emulsion or as a water-in-oil liquid emulsion. In additionto the common dosage forms set out above, the compounds of theinvention, and/or pharmaceutically acceptable salt(s) thereof, can alsobe administered by controlled release means and/or delivery devices. Thecompositions can be prepared by any of the methods of pharmacy. Ingeneral, such methods include a step of bringing into association theactive ingredient with the carrier that constitutes one or morenecessary ingredients. In general, the compositions are prepared byuniformly and intimately admixing the active ingredient with liquidcarriers or finely divided solid carriers or both. The product can thenbe conveniently shaped into the desired presentation.

Thus, the pharmaceutical compositions of this invention can include apharmaceutically acceptable carrier and a compound or a pharmaceuticallyacceptable salt of the compounds of the invention. The compounds of theinvention, or pharmaceutically acceptable salts thereof, can also beincluded in pharmaceutical compositions in combination with one or moreother therapeutically active compounds.

The pharmaceutical carrier employed can be, for example, a solid,liquid, or gas. Examples of solid carriers include lactose, terra alba,sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, andstearic acid. Examples of liquid carriers are sugar syrup, peanut oil,olive oil, and water. Examples of gaseous carriers include carbondioxide and nitrogen.

In preparing the compositions for oral dosage form, any convenientpharmaceutical media can be employed. For example, water, glycols, oils,alcohols, flavoring agents, preservatives, coloring agents and the likecan be used to form oral liquid preparations such as suspensions,elixirs and solutions; while carriers such as starches, sugars,microcrystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents, and the like can be used to form oralsolid preparations such as powders, capsules and tablets. Because oftheir ease of administration, tablets and capsules are the preferredoral dosage units whereby solid pharmaceutical carriers are employed.Optionally, tablets can be coated by standard aqueous or nonaqueoustechniques

A tablet containing the composition of this invention can be prepared bycompression or molding, optionally with one or more accessoryingredients or adjuvants. Compressed tablets can be prepared bycompressing, in a suitable machine, the active ingredient in afree-flowing form such as powder or granules, optionally mixed with abinder, lubricant, inert diluent, surface active or dispersing agent.Molded tablets can be made by molding in a suitable machine, a mixtureof the powdered compound moistened with an inert liquid diluent.

The pharmaceutical compositions of the present invention comprise acompound of the invention (or pharmaceutically acceptable salts thereof)as an active ingredient, a pharmaceutically acceptable carrier, andoptionally one or more additional therapeutic agents or adjuvants. Theinstant compositions include compositions suitable for oral, rectal,topical, and parenteral (including subcutaneous, intramuscular, andintravenous) administration, although the most suitable route in anygiven case will depend on the particular host, and nature and severityof the conditions for which the active ingredient is being administered.The pharmaceutical compositions can be conveniently presented in unitdosage form and prepared by any of the methods well known in the art ofpharmacy.

Pharmaceutical compositions of the present invention suitable forparenteral administration can be prepared as solutions or suspensions ofthe active compounds in water. A suitable surfactant can be includedsuch as, for example, hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofin oils. Further, a preservative can be included to prevent thedetrimental growth of microorganisms.

Pharmaceutical compositions of the present invention suitable forinjectable use include sterile aqueous solutions or dispersions.Furthermore, the compositions can be in the form of sterile powders forthe extemporaneous preparation of such sterile injectable solutions ordispersions. In all cases, the final injectable form must be sterile andmust be effectively fluid for easy syringability. The pharmaceuticalcompositions must be stable under the conditions of manufacture andstorage; thus, preferably should be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol and liquid polyethyleneglycol), vegetable oils, and suitable mixtures thereof.

Pharmaceutical compositions of the present invention can be in a formsuitable for topical use such as, for example, an aerosol, cream,ointment, lotion, dusting powder, mouth washes, gargles, and the like.Further, the compositions can be in a form suitable for use intransdermal devices. These formulations can be prepared, utilizing acompound of the invention, or pharmaceutically acceptable salts thereof,via conventional processing methods. As an example, a cream or ointmentis prepared by mixing hydrophilic material and water, together withabout 5 wt % to about 10 wt % of the compound, to produce a cream orointment having a desired consistency.

Pharmaceutical compositions of this invention can be in a form suitablefor rectal administration wherein the carrier is a solid. It ispreferable that the mixture forms unit dose suppositories. Suitablecarriers include cocoa butter and other materials commonly used in theart. The suppositories can be conveniently formed by first admixing thecomposition with the softened or melted carrier(s) followed by chillingand shaping in moulds.

In addition to the aforementioned carrier ingredients, thepharmaceutical formulations described above can include, as appropriate,one or more additional carrier ingredients such as diluents, buffers,flavoring agents, binders, surface-active agents, thickeners,lubricants, preservatives (including anti-oxidants) and the like.Furthermore, other adjuvants can be included to render the formulationisotonic with the blood of the intended recipient. Compositionscontaining a compound of the invention, and/or pharmaceuticallyacceptable salts thereof; can also be prepared in powder or liquidconcentrate form.

In the treatment conditions which require inhibition or negativemodulation of ENPP1 protein activity an appropriate dosage level willgenerally be about 0.01 to 500 mg per kg patient body weight per day andcan be administered in single or multiple doses. Preferably, the dosagelevel will be about 0.1 to about 250 mg/kg per day; more preferably 0.5to 100 mg/kg per day. A suitable dosage level can be about 0.01 to 250mg/kg per day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kgper day. Within this range the dosage can be 0.05 to 0.5, 0.5 to 5.0 or5.0 to 50 mg/kg per day. For oral administration, the compositions arepreferably provided in the from of tablets containing 1.0 to 1000milligrams of the active ingredient, particularly 1.0, 5.0, 10, 15, 20,25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900 and1000 milligrams of the active ingredient for the symptomatic adjustmentof the dosage of the patient to be treated. The compound can beadministered on a regimen of 1 to 4 times per day, preferably once ortwice per day. This dosing regimen can be adjusted to provide theoptimal therapeutic response.

It is understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors. Such factorsinclude the age, body weight, general health, sex, and diet of thepatient. Other factors include the time and route of administration,rate of excretion, drug combination, and the type and severity of theparticular disease undergoing therapy.

The present invention is further directed to a method for themanufacture of a medicament for inhibiting or negatively modulatingENPP1 protein activity (e.g., treatment of a disorder of uncontrolledcellular proliferation, or one or more neurodegenerative disordersassociated with ENPP1 dysfunction) in mammals (e.g., humans) comprisingcombining one or more disclosed compounds, products, or compositionswith a pharmaceutically acceptable carrier or diluent. Thus, in oneaspect, the invention relates to a method for manufacturing a medicamentcomprising combining at least one disclosed compound or at least onedisclosed product with a pharmaceutically acceptable carrier or diluent.

The disclosed pharmaceutical compositions can further comprise othertherapeutically active compounds, which are usually applied in thetreatment of the above mentioned pathological conditions.

It is understood that the disclosed compositions can be prepared fromthe disclosed compounds. It is also understood that the disclosedcompositions can be employed in the disclosed methods of using.

E. Methods of Using the Compounds and Compositions

The disclosed compounds can be used as single agents or in combinationwith one or more other drugs in the treatment, prevention, control,amelioration or reduction of risk of the aforementioned diseases,disorders and conditions for which compounds of formula I or the otherdrugs have utility, where the combination of drugs together are safer ormore effective than either drug alone. The other drug(s) can beadministered by a route and in an amount commonly used therefore,contemporaneously or sequentially with a disclosed compound. When adisclosed compound is used contemporaneously with one or more otherdrugs, a pharmaceutical composition in unit dosage form containing suchdrugs and the disclosed compound is preferred. However, the combinationtherapy can also be administered on overlapping schedules. It is alsoenvisioned that the combination of one or more active ingredients and adisclosed compound will be more efficacious than either as a singleagent.

The pharmaceutical compositions and methods of the present invention canfurther comprise other therapeutically active compounds as noted hereinwhich are usually applied in the treatment of the above mentionedpathological conditions.

1. Treatment Methods

The compounds disclosed herein are useful for treating, preventing,ameliorating, controlling or reducing the risk of a variety of disorderswherein the patient or subject would benefit from inhibition or negativemodulation of ENPP1. In one aspect, provided is a method of treating orpreventing a disorder in a subject comprising the step of administeringto the subject at least one disclosed compound; at least one disclosedpharmaceutical composition; and/or at least one disclosed product in adosage and amount effective to treat the disorder in the subject.

Also provided is a method for the treatment of one or more disorders,for which ENPP1 inhibition is predicted to be beneficial, in a subjectcomprising the step of administering to the subject at least onedisclosed compound; at least one disclosed pharmaceutical composition;and/or at least one disclosed product in a dosage and amount effectiveto treat the disorder in the subject.

In one aspect, provided is a method for treating a disorder ofuncontrolled cellular proliferation, comprising: administering to asubject at least one disclosed compound; at least one disclosedpharmaceutical composition; and/or at least one disclosed product in adosage and amount effective to treat the disorder in the subject. In afurther aspect, provided is a method for treating or preventing aneurodegenerative disorder, comprising: administering to a subject atleast one disclosed compound; at least one disclosed pharmaceuticalcomposition; and/or at least one disclosed product in a dosage andamount effective to treat the disorder in the subject. Also provided isa method for the treatment of a disorder in a mammal comprising the stepof administering to the mammal at least one disclosed compound,composition, or medicament.

The invention is directed at the use of described chemical compositionsto treat diseases or disorders in patients (preferably human) whereinwherein ENPP1 inhibition would be predicted to have a therapeuticeffect, such as disorders of uncontrolled cellular proliferation (e.g.cancers) and neurodegenerative disorders such as Alzheimer's disease,Huntington's disease, and Parkinson's disease, diseases caused bybacteria and/or viruses, by administering one or more disclosedcompounds or products.

The compounds of the invention can also be used for immunotherapy. Inone embodiment, the compounds of the invention treat disorders ofuncontrolled cellular proliferation, and/or diseases caused by bacteriaand/or viruses through immunotherapy, meaning that the compounds elicitimmunotherapeutic response which results in the treatment of thesediseases.

The compounds disclosed herein are useful for treating, preventing,ameliorating, controlling or reducing the risk of a variety of disordersof uncontrolled cellular proliferation.

Also provided is a method of use of a disclosed compound, composition,or medicament. In one aspect, the method of use is directed to thetreatment of a disorder. In a further aspect, the disclosed compoundscan be used as single agents or in combination with one or more otherdrugs in the treatment, prevention, control, amelioration or reductionof risk of the aforementioned diseases, disorders and conditions forwhich the compound or the other drugs have utility, where thecombination of drugs together are safer or more effective than eitherdrug alone. The other drug(s) can be administered by a route and in anamount commonly used therefore, contemporaneously or sequentially with adisclosed compound. When a disclosed compound is used contemporaneouslywith one or more other drugs, a pharmaceutical composition in unitdosage form containing such drugs and the disclosed compound ispreferred. However, the combination therapy can also be administered onoverlapping schedules. It is also envisioned that the combination of oneor more active ingredients and a disclosed compound can be moreefficacious than either as a single agent.

Examples of disorders treatable with the provided compounds include adisorder of uncontrolled cellular proliferation. In a yet furtheraspect, the disorder of uncontrolled cellular proliferation is cancer.In a yet further aspect, the cancer is a leukemia. In an even furtheraspect, the cancer is a sarcoma. In a still further aspect, the canceris a solid tumor. In a yet further aspect, the cancer is a lymphoma.

It is understood that cancer refer to or describe the physiologicalcondition in mammals that is typically characterized by unregulated cellgrowth. The cancer may be multi-drug resistant (MDR) or drug-sensitive.Examples of cancer include but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, and leukemia. More particular examples of suchcancers include breast cancer, prostate cancer, colon cancer, squamouscell cancer, small-cell lung cancer, non-small cell lung cancer,gastrointestinal cancer, pancreatic cancer, cervical cancer, ovariancancer, peritoneal cancer, liver cancer, e.g., hepatic carcinoma,bladder cancer, colorectal cancer, endometrial carcinoma, kidney cancer,and thyroid cancer.

In various aspects, further examples of cancers are basal cellcarcinoma, biliary tract cancer; bone cancer; brain and CNS cancer;choriocarcinoma; connective tissue cancer; esophageal cancer; eyecancer; cancer of the head and neck; gastric cancer; intra-epithelialneoplasm; larynx cancer; lymphoma including Hodgkin's and Non-Hodgkin'slymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g.,lip, tongue, mouth, and pharynx); retinoblastoma; rhabdomyosarcoma;rectal cancer; cancer of the respiratory system; sarcoma; skin cancer;stomach cancer; testicular cancer; uterine cancer; cancer of the urinarysystem, as well as other carcinomas and sarcomas

In a further aspect, the cancer is a hematological cancer. In a stillfurther aspect, the hematological cancer is selected from acute myeloidleukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloidleukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia,chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia(JMML), Hodgkin lymphoma, Non-Hodgkin lymphoma, multiple myeloma,solitary myeloma, localized myeloma, and extramedullary myeloma. In astill further aspect, the cancer is selected from chronic lymphocyticleukemia, small lymphocytic lymphoma, B-cell non-Hodgkin lymphoma, andlarge B-cell lymphoma.

In a further aspect, the cancer is a cancer of the brain. In a stillfurther aspect, the cancer of the brain is selected from a glioma,medulloblastoma, primitive neuroectodermal tumor (PNET), acousticneuroma, glioma, meningioma, pituitary adenoma, schwannoma, CNSlymphoma, primitive neuroectodermal tumor, craniopharyngioma, chordoma,medulloblastoma, cerebral neuroblastoma, central neurocytoma,pineocytoma, pineoblastoma, atypical teratoid rhabdoid tumor,chondrosarcoma, chondroma, choroid plexus carcinoma, choroid plexuspapilloma, craniopharyngioma, dysembryoplastic neuroepithelial tumor,gangliocytoma, germinoma, hemangioblastoma, hemangiopercytoma, andmetastatic brain tumor. In a yet further aspect, the glioma is selectedfrom ependymoma, astrocytoma, oligodendroglioma, and oligoastrocytoma.In an even further aspect, the glioma is selected from juvenilepilocytic astrocytoma, subependymal giant cell astrocytoma,ganglioglioma, subependymoma, pleomorphic xanthoastrocytom, anaplasticastrocytoma, glioblastoma multiforme, brain stem glioma,oligodendroglioma, ependymoma, oligoastrocytoma, cerebellar astrocytoma,desmoplastic infantile astrocytoma, subependymal giant cell astrocytoma,diffuse astrocytoma, mixed glioma, optic glioma, gliomatosis cerebri,multifocal gliomatous tumor, multicentric glioblastoma multiforme tumor,paraganglioma, and ganglioglioma.

In one aspect, the cancer can be a cancer selected from cancers of theblood, brain, genitourinary tract, gastrointestinal tract, colon,rectum, breast, kidney, lymphatic system, stomach, lung, pancreas, andskin. In a further aspect, the cancer is selected from prostate cancer,glioblastoma multiforme, endometrial cancer, breast cancer, and coloncancer. In a further aspect, the cancer is selected from a cancer of thebreast, ovary, prostate, head, neck, and kidney. In a still furtheraspect, the cancer is selected from cancers of the blood, brain,genitourinary tract, gastrointestinal tract, colon, rectum, breast,livery, kidney, lymphatic system, stomach, lung, pancreas, and skin. Ina yet further aspect, the cancer is selected from a cancer of the lungand liver. In an even further aspect, the cancer is selected from acancer of the breast, ovary, testes and prostate. In a still furtheraspect, the cancer is a cancer of the breast. In a yet further aspect,the cancer is a cancer of the ovary. In an even further aspect, thecancer is a cancer of the prostate. In a still further aspect, thecancer is a cancer of the testes.

In various aspects, disorders associated with an ENPP1 dysfunctioninclude neurodegenerative disorders. In a further aspect, theneurodegenerative disease is selected from Alzheimer's disease,Parkinson's disease, and Huntington's disease.

The compounds are further useful in a method for the prevention,treatment, control, amelioration, or reduction of risk of the diseases,disorders and conditions noted herein. The compounds are further usefulin a method for the prevention, treatment, control, amelioration, orreduction of risk of the aforementioned diseases, disorders andconditions in combination with other agents.

The present invention is further directed to administration of an ENPP1inhibitor for improving treatment outcomes in the context of disordersof uncontrolled cellular proliferation, including cancer. That is, inone aspect, the invention relates to a cotherapeutic method comprisingthe step of administering to a mammal an effective amount and dosage ofat least one compound of the invention in connection with cancertherapy.

In a further aspect, administration improves treatment outcomes in thecontext of cancer therapy. Administration in connection with cancertherapy can be continuous or intermittent. Administration need not besimultaneous with therapy and can be before, during, and/or aftertherapy. For example, cancer therapy can be provided within 1, 2, 3, 4,5, 6, 7 days before or after administration of the compound. As afurther example, cancer therapy can be provided within 1, 2, 3, or 4weeks before or after administration of the compound. As a still furtherexample, cognitive or behavioral therapy can be provided before or afteradministration within a period of time of 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 half-lives of the administered compound.

In one aspect, the disclosed compounds can be used in combination withone or more other drugs in the treatment, prevention, control,amelioration, or reduction of risk of diseases or conditions for whichdisclosed compounds or the other drugs can have utility, where thecombination of the drugs together are safer or more effective thaneither drug alone. Such other drug(s) can be administered, by a routeand in an amount commonly used therefor, contemporaneously orsequentially with a compound of the present invention. When a compoundof the present invention is used contemporaneously with one or moreother drugs, a pharmaceutical composition in unit dosage form containingsuch other drugs and a disclosed compound is preferred. However, thecombination therapy can also include therapies in which a disclosedcompound and one or more other drugs are administered on differentoverlapping schedules. It is also contemplated that when used incombination with one or more other active ingredients, the disclosedcompounds and the other active ingredients can be used in lower dosesthan when each is used singly.

Accordingly, the pharmaceutical compositions include those that containone or more other active ingredients, in addition to a compound of thepresent invention.

The above combinations include combinations of a disclosed compound notonly with one other active compound, but also with two or more otheractive compounds. Likewise, disclosed compounds can be used incombination with other drugs that are used in the prevention, treatment,control, amelioration, or reduction of risk of the diseases orconditions for which disclosed compounds are useful. Such other drugscan be administered, by a route and in an amount commonly used therefor,contemporaneously or sequentially with a compound of the presentinvention. When a compound of the present invention is usedcontemporaneously with one or more other drugs, a pharmaceuticalcomposition containing such other drugs in addition to a disclosedcompound is preferred. Accordingly, the pharmaceutical compositionsinclude those that also contain one or more other active ingredients, inaddition to a compound of the present invention.

The weight ratio of a disclosed compound to the second active ingredientcan be varied and will depend upon the effective dose of eachingredient. Generally, an effective dose of each will be used. Thus, forexample, when a compound of the present invention is combined withanother agent, the weight ratio of a disclosed compound to the otheragent will generally range from about 1000:1 to about 1:1000, preferablyabout 200:1 to about 1:200. Combinations of a compound of the presentinvention and other active ingredients will generally also be within theaforementioned range, but in each case, an effective dose of each activeingredient should be used.

In such combinations a disclosed compound and other active agents can beadministered separately or in conjunction. In addition, theadministration of one element can be prior to, concurrent to, orsubsequent to the administration of other agent(s).

Accordingly, the subject compounds can be used alone or in combinationwith other agents which are known to be beneficial in the subjectindications or other drugs that affect receptors or enzymes that eitherincrease the efficacy, safety, convenience, or reduce unwanted sideeffects or toxicity of the disclosed compounds. The subject compound andthe other agent can be coadministered, either in concomitant therapy orin a fixed combination.

In one aspect, the compound can be employed in combination withanti-cancer therapeutic agents or other known therapeutic agents.

In the treatment of conditions which require inhibition or negativemodulation of ENPP1, an appropriate dosage level will generally be about0.01 to 1000 mg per kg patient body weight per day which can beadministered in single or multiple doses. Preferably, the dosage levelwill be about 0.1 to about 250 mg/kg per day; more preferably about 0.5to about 100 mg/kg per day. A suitable dosage level can be about 0.01 to250 mg/kg per day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50mg/kg per day. Within this range the dosage can be 0.05 to 0.5, 0.5 to 5or 5 to 50 mg/kg per day. For oral administration, the compositions arepreferably provided in the form of tablets containing 1.0 to 1000milligrams of the active ingredient, particularly 1.0, 5.0, 10, 15, 20,25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, and1000 milligrams of the active ingredient for the symptomatic adjustmentof the dosage to the patient to be treated. The compounds can beadministered on a regimen of 1 to 4 times per day, preferably once ortwice per day. This dosage regimen can be adjusted to provide theoptimal therapeutic response. It will be understood, however, that thespecific dose level and frequency of dosage for any particular patientcan be varied and will depend upon a variety of factors including theactivity of the specific compound employed, the metabolic stability andlength of action of that compound, the age, body weight, general health,sex, diet, mode and time of administration, rate of excretion, drugcombination, the severity of the particular condition, and the hostundergoing therapy.

Thus, in one aspect, the invention relates to methods for inhibiting ornegatively modulating ENPP1 in at least one cell, comprising the step ofcontacting the at least one cell with at least one compound of theinvention, in an amount effective to modulate or activate ENPP1 activityresponse, e.g. in the at least one cell. In a further aspect, the cellis mammalian, for example human. In a further aspect, the cell has beenisolated from a subject prior to the contacting step. In a furtheraspect, contacting is via administration to a subject.

a. Treatment of a Disorder of Uncontrolled Cellular Proliferation

In one aspect, the invention relates to a method for the treatment of adisorder of uncontrolled cellular proliferation in a mammal, the methodcomprising the step of administering to the mammal an effective amountof least one disclosed compound or a product of a disclosed method ofmaking a compound, or a pharmaceutically acceptable salt, hydrate,solvate, or polymorph thereof, thereby treating the disorder ofuncontrolled cellular proliferation.

In a still further aspect, the effective amount is a therapeuticallyeffective amount. In a yet still further aspect, the effective amount isa prophylactically effective amount.

In a further aspect, the mammal is a human. In a yet further aspect, themethod further comprises the step of identifying a mammal in need oftreatment of a disorder of uncontrolled cellular proliferation. In astill further aspect, the mammal has been diagnosed with a need fortreatment of a disorder of uncontrolled cellular proliferation prior tothe administering step.

In a further aspect, the disorder of uncontrolled cellular proliferationis a cancer. In a yet further aspect, the cancer is a leukemia. In aneven further aspect, the cancer is a sarcoma. In a still further aspect,the cancer is a solid tumor. In a yet further aspect, the cancer is alymphoma. In an even further aspect, the cancer is selected from chroniclymphocytic leukemia, small lymphocytic lymphoma, B-cell non-Hodgkinlymphoma, and large B-cell lymphoma. In a still further aspect, thecancer is selected from cancers of the blood, brain, genitourinarytract, gastrointestinal tract, colon, rectum, breast, livery, kidney,lymphatic system, stomach, lung, pancreas, and skin. In a yet furtheraspect, the cancer is selected from a cancer of the lung and liver. Inan even further aspect, the cancer is selected from a cancer of thebreast, ovary, testes and prostate. In a still further aspect, thecancer is a cancer of the breast. In a yet further aspect, the cancer isa cancer of the ovary. In an even further aspect, the cancer is a cancerof the prostate. In a still further aspect, the cancer is a cancer ofthe testes.

EXAMPLES F. Experimental

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure accuracywith respect to numbers (e.g., amounts, temperature, etc.), but someerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

Several methods for preparing the compounds of this invention areillustrated in the following Examples. Starting materials and therequisite intermediates are in some cases commercially available, or canbe prepared according to literature procedures or as illustrated herein.

The following exemplary compounds of the invention were synthesized. TheExamples are provided herein to illustrate the invention, and should notbe construed as limiting the invention in any way. The Examples aretypically depicted in free base form, according to the IUPAC namingconvention. However, some of the Examples were obtained or isolated insalt form.

Some of the Examples were obtained as racemic mixtures of one or moreenantiomers or diastereomers. The compounds may be separated by oneskilled in the art to isolate individual enantiomers. Separation can becarried out by the coupling of a racemic mixture of compounds to anenantiomerically pure compound to form a diastereomeric mixture,followed by separation of the individual diastereomers by standardmethods, such as fractional crystallization or chromatography. A racemicor diastereomeric mixture of the compounds can also be separateddirectly by chromatographic methods using chiral stationary phases.

Experimental Chemistry Synthesis Schemes, Methods and Procedures6-Chloro-3H-imidazo[4,5-c]pyridine-2-thiol (Compound 2)

The reaction mixture of 6-chloropyridine-3,4-diamine (100 mg, 0.697mmol), di(1H-imidazol-1-yl)methanethione (124 mg, 0.697 mmol) intetrahydrofuran (4 mL, 0.697 mmol) was stirred at RT overnight. Theprecipitate was filtrated and 6-chloro-3H-imidazo[4,5-c]pyridine-2-thiol(100 mg, 0.539 mmol, 77% yield) was obtained. ¹HNMR (400 MHz, CD₃OD) δ7.76 (s, 1H), 7.07 (s, 1H), ESI-MS: m/z 185.82 (M+H)³⁰

2-Chloro-N-(3,4-dimethoxyphenyl)acetamide (Compound 1)

To the solution of 3,4-dimethoxyaniline (200 mg, 1.306 mmol), TEA (0.546mL, 3.92 mmol) in DCM (5 mL) was added 2-chloroacetyl chloride (0.125mL, 1.567 mmol) at room temperature. After 1 hr, concentration andpurification on combiflash (4 g, EtOAc/Hexane) gave2-chloro-N-(3,4-dimethoxyphenyl)acetamide (232 mg, 1.010 mmol, 77%yield). 1HNMR (400 MHz, CDCl3) δ 7.28 (J=2 Hz, 1H), 6.95 (d, J=6 Hz,1H), 6.85 (d, J=8.8 Hz, 1H), 4.19 (s, 2H) 3.90 (s, 3H), 3.87 (s, 3H).

2-((6-Chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3,4-dimethoxyphenyl)acetamide(Compound 3)

The reaction mixture of 6-chloro-3H-imidazo[4,5-c]pyridine-2-thiol (13mg, 0.070 mmol), 2-chloro-N-(3,4-dimethoxyphenyl)acetamide (16.08 mg,0.070 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.029 mL,0.210 mmol) in acetonitrile (1 mL) was heated at 65° C. for 1.5 hr.Concentration and purification on combiflash (4 g, MeOH/DCM) gave2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3,4-dimethoxyphenyl)acetamide(13 mg, 0.034 mmol, 49.0% yield). 1HNMR (400 MHz, CDCl₃) δ 9.81 (s, 1H),8.70 (m, 1H), 7.31 (s, 1H), 6.86 (m, 1H), 6.77 (m, 2H), 3.99 (s, 2H),3.84 (m, 6H). ESI-MS: m/z 379.02 (M+H)+

N-(3,4-dimethoxyphenyl)-2-((5-methoxythiazolo[4,5-b]pyridin-2-yl)thio)acetamide(Compound 4)

The reaction mixture of 5-methoxythiazolo[4,5-b]pyridine-2-thiol (17.27mg, 0.087 mmol), 2-chloro-N-(3,4-dimethoxyphenyl)acetamide (20 mg, 0.087mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.036 mL, 0.261mmol) in acetonitrile (1 mL) was heated at 65° C. for 2 hr.Concentration and purification on combiflash (4 g, MeOH/DCM) gaveN-(3,4-dimethoxyphenyl)-2-((5-methoxythiazolo[4,5-b]pyridin-2-yl)thio)acetamide(13.5 mg, 0.034 mmol, 39.6% yield) ¹HNMR (400 MHz, CD₃OD), δ 8.01 (d,J=8.8 Hz, 1H), 7.82 (s, 1H), 7.31 (d, J=2 Hz, 1H), 7.02 (d, J=8.8 Hz,1H), 6.86 (m, 2H), 3.95 (s, 2H), 3.80 (s, 3H), 3.30 (m, 6H). ESI-MS: m/z392.04 (M+H)⁺

N-(benzo[d][1,3]dioxol-5-yl)-2-chloroacetamide (Compound 5)

To the solution of benzo[d][1,3]dioxol-5-amine (200 mg, 1.458 mmol), TEA(0.610 mL, 4.38 mmol) in DCM (5 mL) was added 2-chloroacetyl chloride(0.139 mL, 1.75 mmol) at room temperature. After 1 hr, concentration andpurification on combiflash (4 g, EtOAc/Hexane) gaveN-(benzo[d][1,3]dioxol-5-yl)-2-chloroacetamide (239 mg, 1.119 mmol, 77%yield).

¹HNMR (400 MHz, CDCl₃) δ 8.12 (br-s, 1H), 7.23 (s, 1H), 6.85 (d, J=8 Hz,1H), 6.78 (d, J=8 Hz, 1H), 5.97 (s, 2H), 4.18 (s, 2H) ESI-MS: m/z 213.96(M+H)⁺

N-(benzo[d][1,3]dioxol-5-yl)-2-((5-methoxythiazolo[4,5-b]pyridin-2-yl)thio)acetamide(Compound 6)

The reaction mixture of 5-methoxythiazolo[4,5-b]pyridine-2-thiol (18.56mg, 0.094 mmol), N-(benzo[d][1,3]dioxol-5-yl)-2-chloroacetamide (20 mg,0.094 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.039 mL,0.281 mmol) in Acetonitrile (1 mL) was heated at 65° C. for 1.5 hr.Concentration and purification on combiflash (4 g, MeOH/DCM) gaveN-(benzo[d][1,3]dioxol-5-yl)-2-((5-methoxythiazolo[4,5-b]pyridin-2-yl)thio)acetamide(17.8 mg, 0.047 mmol, 50.6% yield) ¹HNMR (400 MHz, CDCl₃) δ 9.63 (s,1H), 8.01 (d, J=8.8 Hz, 2H), 7.22 (s, 1H), 6.86 (d, J=8.8 Hz, 1H), 6.72(m, 3H), 5.93 (s, 2H), 4.02 (s, 2H), 3.99 (s, 3H), ESI-MS: m/z 376.0(M+H)⁺

N-(benzo[d][1,3]dioxol-5-yl)-2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide(Compound 7)

The reaction mixture of 6-chloro-1H-imidazo[4,5-c]pyridine-2-thiol(17.38 mg, 0.094 mmol), N-(benzo[d][1,3]dioxol-5-yl)-2-chloroacetamide(20 mg, 0.094 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine(0.039 mL, 0.281 mmol) in acetonitrile (1 mL) was heated at 65° C. for1.5 hr. Concentration and purification on combiflash (4 g, MeOH/DCM)gaveN-(benzo[d][1,3]dioxol-5-yl)-2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide(11.8 mg, 0.033 mmol, 34.7% yield). ¹HNMR (400 MHz, CDCl₃) δ 8.44 (s,1H), 7.34 (s, 1H), 7.13 (s, 1H), 6.75 (d, J=8.4 Hz, 1H), 6.64 (d, J=8.0Hz 1H), 5.84 (s, 2H), 3.94 (s, 2H), ESI-MS: m/z 362.98 (M+H)⁺

N-(3,4-dimethoxyphenyl)-2-((7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide(Compound 9)

The reaction mixture of 7-methyl-1H-imidazo[4,5-c]pyridine-2-thiol(14.39 mg, 0.087 mmol), 2-chloro-N-(3,4-dimethoxyphenyl)acetamide (20mg, 0.087 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.036mL, 0.261 mmol) in acetonitrile (1 mL) was heated at 65° C. for 2 hr.Concentration and purification on combiflash (4 g, DCM/MeOH) gaveN-(3,4-dimethoxyphenyl)-2-((7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide(33 mg, 0.092 mmol, 100% yield) ¹HNMR (400 MHz, CDCl₃), δ 10.8 (s, 1H),8.66 (s, 1H), 7.99 (s, 1H), 7.35 (s, 1H), 7.18 (s, 2H), 4.11 (s, 2H),3.80 (s, 6H), 2.59 (s, 3H). ESI-MS: m/z 359.05 (M+H)⁺

N-(benzo[d][1,3]dioxol-5-yl)-2-((7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide(Compound 10)

The reaction mixture of 7-methyl-1H-imidazo[4,5-c]pyridine-2-thiol(15.47 mg, 0.094 mmol), N-(benzo[d][1,3]dioxol-5-yl)-2-chloroacetamide(20 mg, 0.094 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine(0.039 mL, 0.281 mmol) in acetonitrile (1 mL) was heated to 65° C. for 2hr. Concentration and purification on combiflash (4 g, DCM/MeOH) gaveN-(benzo[d][1,3]dioxol-5-yl)-2-((7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide(33.7 mg, 0.098 mmol, 100% yield) ¹HNMR (400 MHz, CDCl₃) δ 8.01 (m, 2H),7.09 (m, 3H), 6.69 (m, 1H), 5.88 (s, 2H), 3.94 (s, 2H). 2.52 (s, 3H).ESI-MS: m/z 342.98 (M+H)⁺

6-Chlorothiazolo[4,5-c]pyridine-2-thiol (Compound 11)

The reaction mixture of 4,6-dichloropyridin-3-amine (50 mg, 0.307 mmol),potassium O-ethyl carbonodithioate (73.8 mg, 0.460 mmol) in NMP (2 mL)was heated to 150° C. overnight. Acetice acid (60 uL) and water wereadded. Precipitate was filtered and dried to gave6-chlorothiazolo[4,5-c]pyridine-2-thiol (52 mg, 0.257 mmol, 84% yield)¹HNMR (400 MHz, CD₃OD) δ 8.35 (s, 1H), 7.44 (s, 1H). ESI-MS: m/z 202.78(M+H)⁺

2-((6-Chlorothiazolo[4,5-c]pyridin-2-yl)thio)-N-(3,4-dimethoxyphenyl)acetamide(Compound 12)

The reaction mixture of 6-chlorothiazolo[4,5-c]pyridine-2-thiol (17.65mg, 0.087 mmol), 2-chloro-N-(3,4-dimethoxyphenyl)acetamide (20 mg, 0.087mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.036 mL, 0.261mmol) in acetonitrile (1 mL) was heated at 65° C. for 1.5 hr.Concentration and purification by Prep-TLC (MeOH/DCM) gave2-((6-chlorothiazolo[4,5-c]pyridin-2-yl)thio)-N-(3,4-dimethoxyphenyl)acetamide(5.7 mg, 0.014 mmol, 16.53% yield)

¹HNMR (400 MHz, CDCl₃) δ 8.97 (s, 1H), 7.78 (s, 1H), 7.34 (s, 1H), 6.79(m, 2H), 4.11 (s, 2H), 3.86 (s, 3H), 3.84 (s, 3H). ESI-MS: m/z 395.97(M+H)⁺

2-((6-Chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3,4-dimethoxyphenyl)propanamide(Compound 14)

The reaction mixture of 6-chloro-1H-imidazo[4,5-c]pyridine-2-thiol(15.24 mg, 0.082 mmol), 2-chloro-N-(3,4-dimethoxyphenyl)propanamide (20mg, 0.082 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.034mL, 0.246 mmol) in acetonitrile (1 mL) was heated at 65° C. overnight.The solid was filtered and dried to give2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3,4-dimethoxyphenyl)propanamide(17 mg, 0.043 mmol, 52.7% yield). ESI-MS: m/z 393.06 (M+H)⁺

7-Methyl-1,3-dihydro-2H-imidazo[4,5-c]pyridine-2-thione (Compound 8)

To a solution of 5-methylpyridine-3,4-diamine (100 mg, 0.812 mmol) andN,N-dimethylpyridin-4-amine (109 mg, 0.893 mmol) in MeCN was addeddi(1H-imidazol-1-yl)methanethione (217 mg, 1.218 mmol) portionwise. Themixture was stirred at room temperature for 4 h. The precipitate formedwas filtered, washed with water, Et₂O, DCM and dried to give7-methyl-1H-imidazo[4,5-c]pyridine-2 (3H)-thione (100 mg, 75% yield) asa pale red solid. ¹H-NMR (400 MHz, DMSO-d₆) δ ppm 12.73 (brs, 2H), 8.20(s, 1H), 8.06 (s, 1H), 2.33 (s, 3H); ESI-MS: m/z 165.84 (M+H)⁺.

2-Chloro-N-(3-hydroxy-4-methoxyphenyl)acetamide (Compound 15)

A solution of 5-amino-2-methoxyphenol (100 mg, 0.719 mmol) andtriethylamine (0.150 ml, 1.078 mmol) in DCM was cooled to 0° C. and2-chloroacetyl chloride (0.060 ml, 0.755 mmol) was added dropwise. Thereaction mixture was stirred at room temperature for 2 h. Solvent wasremoved and the crude was purified by combiflash SiO₂ chromatography(0-5% MeOH-DCM) to give 2-chloro-N-(3-hydroxy-4-methoxyphenyl)acetamide(58 mg, 37%) as a brown solid. ¹H-NMR (400 MHz, CDCl₃) δ ppm 8.11 (brs,1H), 7.14 (d, 1H, J=4 Hz), 7.04 (dd, 1H, J=4 Hz and J=8 Hz), 6.81 (d,1H, J=12 Hz), 4.18 (s, 2H), 3.88 (s, 3H); ESI-MS: m/z 215.90 (M+H)⁺.

N-(3-hydroxy-4-methoxyphenyl)-2-((7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide(Compound 16)

A mixture of 2-chloro-N-(3-hydroxy-4-methoxyphenyl)acetamide (15 mg,0.070 mmol), 7-methyl-1H-imidazo[4,5-c]pyridine-2 (3H)-thione (12.64 mg,0.077 mmol), sodium iodide (10.43 mg, 0.070 mmol) and triethylamine(0.015 ml, 0.104 mmol) in MeCN was heated to 65° C. for 2 h. Solvent wasremoved and the crude was purified by combiflash SiO₂ chromatography(0-10% MeOH-DCM) to obtainN-(3-hydroxy-4-methoxyphenyl)-2-((7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide(11 mg, 46%) as an off-white solid. ¹H-NMR (400 MHz, CD₃OD/CDCl₃) δ ppm8.52 (s, 1H), 7.93 (s, 1H), 7.08 (d, 1H, J=4 Hz), 6.92 (dd, 1H, J=4 Hz,J=8 Hz), 6.77 (d, 1H, J=8 Hz), 4.07 (s, 2H), 3.80 (s, 3H), 2.54 (s, 3H);ESI-MS: m/z 345.03 (M+H)⁺.

The reaction mixture of 7-methyl-1H-imidazo[4,5-c]pyridine-2-thiol(14.39 mg, 0.087 mmol), 2-chloro-N-(3,4-dimethoxyphenyl)acetamide (20mg, 0.087 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.036mL, 0.261 mmol) in acetonitrile (1 mL) was heated at 65° C. for 2 hr.Concentration and purification on combiflash (4 g, DCM/MeOH) gaveN-(3,4-dimethoxyphenyl)-2-((7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide(33 mg, 0.092 mmol, 100% yield) ¹HNMR (400 MHz, CDCl₃), 10.8 (s, 1H),8.66 (s, 1H), 7.99 (s, 1H), 7.35 (s, 1H), 7.18 (s, 2H), 4.11 (s, 2H),3.80 (s, 6H), 2.59 (s, 3H). ESI-MS: m/z 359.05 (M+H)+

2-Chloro-N-(4-hydroxy-3-methoxyphenyl)acetamide (Compound 17)

A solution of 4-amino-2-methoxyphenol (100 mg, 0.719 mmol) andtriethylamine (0.150 ml, 1.078 mmol) in DCM was cooled to 0° C. and2-chloroacetyl chloride (0.060 ml, 0.755 mmol) was added dropwise. Thereaction mixture was stirred at room temperature for 2 h. Solvent wasremoved and the crude was purified by combiflash SiO₂ chromatography(0-5% MeOH-DCM) to give 2-chloro-N-(4-hydroxy-3-methoxyphenyl)acetamide(55 mg, 36% yield) as a brown solid. ¹H-NMR (400 MHz, CDCl₃) δ ppm 8.14(brs, 1H), 7.38 (d, 1H, J=2.4 Hz), 6.87 (d, 1H, J=8 Hz), 6.80 (dd, 1H,J=4 Hz and J=8 Hz), 4.18 (s, 2H), 3.90 (s, 3H); ESI-MS: m/z 215.90(M+H)⁺.

N-(4-hydroxy-3-methoxyphenyl)-2-((7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide(Compound 18)

A mixture of 7-methyl-1H-imidazo[4,5-c]pyridine-2 (3H)-thione (13.79 mg,0.083 mmol), 2-chloro-N-(4-hydroxy-3-methoxyphenyl)acetamide (15 mg,0.070 mmol), Sodium iodide (10.43 mg, 0.070 mmol) in MeCN was addedtriethylamine (0.012 ml, 0.083 mmol) and heated to 65° C. for 1 h.Solvent was removed and the crude was purified by combiflash (0-10%DCM-MeOH) to obtainN-(4-hydroxy-3-methoxyphenyl)-2-((7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide(14 mg, 58%) as a white solid. ¹H-NMR (400 MHz, CD₃OD/CDCl₃) δ ppm 8.51(s, 1H), 7.91 (s, 1H), 7.27 (d, 1H, J=4 Hz), 6.77 (dd, 1H, J=4 Hz, J=8Hz), 6.71 (d, 1H, J=8 Hz), 4.06 (s, 2H), 3.80 (s, 3H), 2.53 (s, 3H);ESI-MS: m/z 345.04 (M+H)⁺.

N-(3-hydroxy-4-methoxyphenyl)-2-((5-methoxythiazolo[4,5-b]pyridin-2-yl)thio)acetamide(Compound 19)

To a mixture of 5-methoxythiazolo[4,5-b]pyridine-2-thiol (20 mg, 0.101mmol), 2-chloro-N-(3-hydroxy-4-methoxyphenyl)acetamide (23.93 mg, 0.111mmol) and sodium iodide (15.02 mg, 0.101 mmol) in MeCN (1 ml) was addedtriethylamine (0.021 ml, 0.151 mmol) and stirred at 65° C. for 2 h.Solvent was removed and the crude was combiflash chromatographed using0-10% MeOH-DCM to obtainN-(3-hydroxy-4-methoxyphenyl)-2-((5-methoxythiazolo[4,5-b]pyridin-2-yl)thio)acetamide(23 mg, 60%) as a pale orange solid. ¹H-NMR (400 MHz, CD₃OD/CDCl₃) δ ppm9.56 (brs, 1H), 7.98 (d, 1H, J=12 Hz), 7.05 (dd, 1H, J=1.2 Hz, J=4 Hz),6.92 (dd, 1H, J=4 Hz, J=8 Hz), 6.83 (d, 1H, J=8 Hz), 6.74 (d, 1H, J=12Hz), 4.00 (s, 2H), 3.96 (s, 3H), 3.81 (s, 3H); ESI-MS: m/z 378.02(M+H)⁺.

N-(4-hydroxy-3-methoxyphenyl)-2-((5-methoxythiazolo[4,5-b]pyridin-2-yl)thio)acetamide(Compound 20)

To a mixture of 2-chloro-N-(4-hydroxy-3-methoxyphenyl)acetamide (20 mg,0.093 mmol), 5-methoxythiazolo[4,5-b]pyridine-2-thiol (20.23 mg, 0.102mmol) and sodium iodide (13.81 mg, 0.093 mmol) in MeCN was addedtriethylamine (0.019 ml, 0.139 mmol) and stirred at 65° C. for 2 h.Solvent was removed and the crude was chromatographed using 0-10%MeOH-DCM to affordN-(4-hydroxy-3-methoxyphenyl)-2-((5-methoxythiazolo[4,5-b]pyridin-2-yl)thio)acetamide(22 mg, 63%) as a pale orange solid. ¹H-NMR (400 MHz, CD₃OD/CDCl₃) δ ppm7.88 (d, 1H, J=12 Hz), 7.22 (d, 1H, J=2 Hz), 6.73 (d, 1H, J=12 Hz), 6.65(d, 1H, J=8 Hz), 6.61 (dd, 1H, J=4 Hz, J=8 Hz), 3.97 (s, 2H), 3.85 (s,3H), 3.72 (s, 3H); ESI-MS: m/z 378.04 (M+H)⁺.

2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-hydroxy-4-methoxyphenyl)acetamide(Compound 21)

A mixture of 2-chloro-N-(3-hydroxy-4-methoxyphenyl)acetamide (15 mg,0.070 mmol), 6-chloro-3H-imidazo[4,5-c]pyridine-2-thiol (15.50 mg, 0.083mmol), sodium iodide (10.43 mg, 0.070 mmol) and triethylamine (0.015 ml,0.104 mmol) in MeCN was heated to 65° C. for 2 h. The crude wassubjected to combiflash silica gel chromatography (0-10% MeOH-DCM) toobtain2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-hydroxy-4-methoxyphenyl)acetamide(15 mg, 59%) as a white solid. 1H-NMR (400 MHz, CD₃OD/CDCl₃) δ ppm 8.49(s, 1H), 7.48 (s, 1H), 7.11 (d, 1H, J=2.4 Hz), 6.94 (dd, 1H, J=4 Hz, J=8Hz), 6.83 (d, 1H, J=8 Hz), 4.18 (s, 2H), 3.81 (s, 3H); ESI-MS: m/z365.02 (M+H)⁺.

2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-hydroxy-3-methoxyphenyl)acetamide(Compound 20)

A mixture of 2-chloro-N-(4-hydroxy-3-methoxyphenyl)acetamide (17.42 mg,0.081 mmol), 6-chloro-3H-imidazo[4,5-c]pyridine-2-thiol (15 mg, 0.081mmol), sodium iodide (12.11 mg, 0.081 mmol) and triethylamine (0.011 ml,0.081 mmol) in MeCN was heated to 65° C. for 2 h. The crude was purifiedby combiflash silica gel chromatography (0-10% MeOH-DCM) to afford2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-hydroxy-3-methoxyphenyl)acetamide(17 mg, 58%) as a white solid. ¹H-NMR (400 MHz, CD₃OD/CDCl₃) δ ppm 8.49(s, 1H), 7.45 (s, 1H), 7.28 (d, 1H, J=2.4 Hz), 6.84 (dd, 1H, J=4 Hz, J=8Hz), 6.73 (d, 1H, J=8 Hz), 4.15 (s, 2H), 3.81 (s, 3H); ESI-MS: m/z365.01 (M+H)⁺.

2-chloro-N-(3,4-dimethoxyphenyl)propanamide (Compound 23)

To a cooled solution of 3,4-dimethoxyaniline (100 mg, 0.653 mmol) andtriethylamine (0.136 ml, 0.979 mmol) in DCM was added 2-chloropropanoylchloride (0.076 ml, 0.783 mmol) and stirred at RT for 12 h. Solvent wasremoved and the crude was purified by combiflash chromatography (0-50%EtOAc-Hexanes) to give 2-chloro-N-(3,4-dimethoxyphenyl)propanamide (140mg, 88%) as a fluffy white solid. ¹H-NMR (400 MHz, CDCl₃) δ ppm 8.19(brs, 1H), 7.33 (d, 1H, J=4 Hz), 6.93 (dd, 1H, J=4 Hz, J=8 Hz), 6.83 (d,1H, J=8 Hz), 4.55 (q, 1H, J=8 Hz), 3.90 (s, 3H), 3.87 (s, 3H), 1.83 (d,3H, J=8 Hz); ESI-MS: m/z 243.90 (M+H)⁺.

2-((6-Chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-hydroxyphenyl)acetamide(Compound 25)

The reaction mixture of 6-chloro-1H-imidazo[4,5-c]pyridine-2-thiol(15.00 mg, 0.081 mmol), 2-chloro-N-(3-hydroxyphenyl)acetamide (15 mg,0.081 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.034 mL,0.242 mmol) in acetonitrile (1 mL) was heated at 65° C. for 2 hr.Concentration and purification on combiflash (4 g, MeOH/DCM) gave2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-hydroxyphenyl)acetamide(5 mg, 0.015 mmol, 18.48% yield)

¹HNMR (400 MHz, CD₃OD) δ 8.49 (s, 1H), 7.47 (s, 1H), 7.13 (m, 2H), 6.96(s, J=8 Hz, 1H), 6.53 (d, J=8 Hz, 1H), 4.19 (s, 1H), ESI-MS: m/z 334.96(M+H)⁺

2-Chloro-N-(3-fluoro-4-methoxyphenyl)acetamide (Compound 26)

To the solution of 3-fluoro-4-methoxyaniline (200 mg, 1.417 mmol) andTEA (0.593 mL, 4.25 mmol) in DCM (5 mL) was added 2-chloroacetylchloride (0.135 mL, 1.700 mmol). After 1 hr, the reaction mixture wasconcentrated and purified on combiflash (12 g, DCM/MeOH) to give2-chloro-N-(3-fluoro-4-methoxyphenyl)acetamide (208 mg, 0.956 mmol,67.5% yield)

¹HNMR (400 MHz, CDCl₃) δ 8.14 (s, 1H), 7.46 (d, J=12.4 Hz, 1H), 7.16 (d,J=8.4 Hz, 1H), 6.93 (m, 1H), 4.18 (s, 2H), 3.88 (s, 3H), ESI-MS: m/z217.88 (M+H)⁺

2-((6-Chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-fluoro-4-methoxyphenyl)acetamide(Compound 27)

The reaction mixture of 6-chloro-1H-imidazo[4,5-c]pyridine-2-thiol(17.06 mg, 0.092 mmol), 2-chloro-N-(3-fluoro-4-methoxyphenyl)acetamide(20 mg, 0.092 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine(0.038 mL, 0.276 mmol) in CH₃CN was heated at 65° C. for 2 hr.Concentration and purification on combiflash (4 g, MeOH/DCM) gave2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-fluoro-4-methoxyphenyl)acetamide(16 mg, 0.044 mmol, 47.5% yield) ¹HNMR (400 MHz, CD₃OD) δ 8.50 (s, 1H),7.48 (m, 2H), 7.18 (d, J=8 Hz, 1H), 7.03 (t, J=8.8 Hz, 1H), 4.22 (s,2H), 3.84 (s, 3H), ESI-MS: m/z 366.97 (M+H)⁺

2-Chloro-N-(4-(difluoromethoxy)-3-methoxyphenyl)acetamide (Compound 28)

To the the reaction mixture of 4-(difluoromethoxy)-3-methoxyaniline (150mg, 0.793 mmol), TEA (0.334 mL, 2.379 mmol) in DCM (4 mL) was added2-chloroacetyl chloride (0.076 mL, 0.952 mmol) dropwise. After 1 hr atrt, the mixture was concentrated and purified on combiflash (12 g,DCM/MeOH) gave 2-chloro-N-(4-(difluoromethoxy)-3-methoxyphenyl)acetamide(134 mg, 0.504 mmol, 63.6% yield) ¹HNMR (400 MHz, CDCl₃) δ 8.23 (s, 1H),7.51 (s, 1H), 7.13 (d, J=8.4 Hz, 1H), 6.92 (m, 1H), 6.7, 6.51 (d, J=75Hz, 1H), 4.2 (s, 2H), 3.89 (s, 3H). ESI-MS: m/z, 265.92 (M+H)⁺

2-((6-Chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-(difluoromethoxy)-3-methoxyphenyl)acetamide(Compound 29)

The reaction mixture of 6-chloro-1H-imidazo[4,5-c]pyridine-2-thiol(13.98 mg, 0.075 mmol),2-chloro-N-(4-(difluoromethoxy)-3-methoxyphenyl)acetamide (20 mg, 0.075mmol), sodium iodide (11.29 mg, 0.075 mmol), triethylamine (0.031 mL,0.226 mmol) in acetonitrile (1 mL) was heated to 65° C. for 2 hr.Concentration and purification by combiflash (4 g, DCM/MeOH) gave2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-(difluoromethoxy)-3-methoxyphenyl)acetamide(24 mg, 0.058 mmol, 77% yield) ¹HNMR (400 MHz, CDCl₃) δ 8.50 (s, 1H),7.56 (s, 1H), 7.38 (s, 1H), 7.02 (d, J=8.4 Hz, 1H), 6.81 (d, J=8.4 Hz,1H), 6.43 (t, J=76 Hz, 1H), 3.99 (s, 1H), 3.80 (s, 1H) ESI-MS: m/z414.97 (M+H)⁺

6-(Trifluoromethyl)-3H-imidazo[4,5-c]pyridine-2-thiol (Compound 30)

The reaction mixture of 6-(trifluoromethyl)pyridine-3,4-diamine (100 mg,0.565 mmol), di(1H-imidazol-1-yl)methanethione (101 mg, 0.565 mmol) intetrahydrofuran (4 mL, 0.565 mmol). Overnight, precipitate was filteredand 6-(trifluoromethyl)-3H-imidazo[4,5-c]pyridine-2-thiol (70 mg, 0.319mmol, 56.6% yield) was obtained. ¹HNMR (400 MHz, CDCl₃—CD₃OD) δ 8.36 (s,1H), 7.42 (s, 1H), ESI: m/z 219.8 (M+H)⁺

N-(3-hydroxy-4-methoxyphenyl)-2-((6-(trifluoromethyl)-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide(Compound 31)

The reaction mixture of6-(trifluoromethyl)-1H-imidazo[4,5-c]pyridine-2-thiol (15.25 mg, 0.070mmol), 2-chloro-N-(3-hydroxy-4-methoxyphenyl)acetamide (15 mg, 0.070mmol), sodium iodide (10.43 mg, 0.070 mmol), triethylamine (0.029 mL,0.209 mmol) in acetonitrile (1 mL) was heated to 65° C. for 2 hr.Concentration and purification by combiflash (4 g, DCM/MeOH) gaveN-(3-hydroxy-4-methoxyphenyl)-2-((6-(trifluoromethyl)-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide(24 mg, 0.060 mmol, 87% yield) ¹HNMR (400 MHz, CDCl₃) δ 9.03 (s, 1H),8.00 (s, 1H), 7.50 (d, J=8.4 Hz, 1H), 7.29 (s, 1H), 7.14 (s, 1H), 6.96(s, 1H), 4.22 (s, 2H), 4.03 (m, 3H). ESI-MS: m/z 399.06 (M+H)⁺

2-chloro-N-(4-hydroxyphenyl)acetamide (Compound 32)

To the the reaction mixture of 4-aminophenol (200 mg, 1.833 mmol), TEA(0.772 mL, 5.50 mmol) in DCM (4 mL) was added 2-chloroacetyl chloride(0.146 mL, 1.833 mmol) dropwise. After 1 hr at rt, the mixture wasconcentrated and purified on combiflash (12 g, DCM/MeOH) gave2-chloro-N-(4-hydroxyphenyl)acetamide (134 mg, 0.722 mmol, 39.4% yield)¹HNMR (400 MHz, CDCl₃) δ 7.32 (d, J=8.8 Hz, 2H), 7.79 (d, J=8.4 Hz, 2H),4.15 (s, 2H) ESI-MS: m/z 185.84 (M+H)+

2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-hydroxyphenyl)acetamide(Compound 33)

The reaction mixture of 6-chloro-1H-imidazo[4,5-c]pyridine-2-thiol(15.00 mg, 0.081 mmol), 2-chloro-N-(4-hydroxyphenyl)acetamide (15 mg,0.081 mmol), sodium iodide (12.11 mg, 0.081 mmol), triethylamine (0.034mL, 0.242 mmol) in Acetonitrile (1 mL) was heated to 65° C. for 2 hr.Concentration and purification by combiflash (4 g, DCM/MeOH) gave2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-hydroxyphenyl)acetamide(24 mg, 0.072 mmol, 89% yield) ¹HNMR (400 MHz, CD₃OD) δ 8.48 (s, 1H),7.38 (s, 1H), 7.30 (d, J=8.8 Hz, 2H) 7.19 (s, 1H), 6.71 (d, J=8.8 Hz,2H), 3.98 (s, 2H), ESI-MS: m/z 334.99 (M+H)⁺

2-chloro-N-(4-methoxy-3-(trifluoromethoxy)phenyl)acetamide (Compound 34)

To the the reaction mixture of 4-methoxy-3-(trifluoromethoxy)aniline(200 mg, 0.965 mmol), TEA (0.407 mL, 2.90 mmol) in DCM (4 mL) was added2-chloroacetyl chloride (0.092 mL, 1.159 mmol) dropwise. After 1 hr atrt, the mixture was concentrated and purified on combiflash (12 g,DCM/MeOH) gave2-chloro-N-(4-methoxy-3-(trifluoromethoxy)phenyl)acetamide (212 mg,0.747 mmol, 77% yield) ¹HNMR (400 MHz, CDCl₃) δ 8.16 (s, 1H), 7.51 (s,1H), 7.43 (d, J=6.4 Hz, 1H), 6.97 (d, J=8.8 Hz, 1H) 4.19 (s, 2H), 3.88(s, 3H). ESI-MS: m/z 283.87 (M+H)⁺

2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-methoxy-3-(trifluoromethoxy)phenyl)acetamide(Compound 35)

The reaction mixture of 6-chloro-1H-imidazo[4,5-c]pyridine-2-thiol (9.82mg, 0.053 mmol),2-chloro-N-(4-methoxy-3-(trifluoromethoxy)phenyl)acetamide (15 mg, 0.053mmol), sodium iodide (7.93 mg, 0.053 mmol), triethylamine (0.022 mL,0.159 mmol) in acetonitrile (1 mL) was heated to 65° C. for 2 hr.Concentration and purification by combiflash (4 g, DCM/MeOH) gave2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-methoxy-3-(trifluoromethoxy)phenyl)acetamide(25 mg, 0.058 mmol, 100% yield) ¹HNMR (400 MHz, CD₃OD) δ 8.50 (s, 1H),7.65 (s, 1H), 7.49 (s, 1H), 7.43 (d, J=6.8 Hz, 1H), 7.10 (d, J=8.8 Hz,1H), 4.23 (s, 2H), 3.85 (s, ESI-MS: m/z 432.97 (M+H)⁺

2-Chloro-N-(4-fluoro-3-hydroxyphenyl)acetamide (Compound 36)

To the the reaction mixture of 5-amino-2-fluorophenol (200 mg, 1.573mmol), TEA (0.663 mL, 4.72 mmol) in DCM (4 mL) was added 2-chloroacetylchloride (0.125 mL, 1.573 mmol) dropwise. After 1 hr at rt, the mixturewas concentrated and purified on combiflash (12 g, DCM/MeOH) gave2-chloro-N-(4-fluoro-3-hydroxyphenyl)acetamide (169 mg, 0.830 mmol,52.8% yield) ¹HNMR (400 MHz, CD₃OD) δ 7.22 (m, 1H), 6.93 (m, 1H), 6.83(m, 1H), 4.07 (s, 2H) ESI-MS: m/z 203.79 (M+H)⁺

2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-hydroxyphenyl)acetamide(Compound 37)

The reaction mixture of 6-chloro-1H-imidazo[4,5-c]pyridine-2-thiol(13.68 mg, 0.074 mmol), 2-chloro-N-(4-fluoro-3-hydroxyphenyl)acetamide(15 mg, 0.074 mmol), sodium iodide (11.04 mg, 0.074 mmol), triethylamine(0.031 mL, 0.221 mmol) in acetonitrile (1 mL) was heated to 65° C. for 2hr. Concentration and purification by combiflash (4 g, DCM/MeOH) gave2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-hydroxyphenyl)acetamide(24 mg, 0.068 mmol, 92% yield) ¹HNMR (400 MHz, CD₃OD) δ 8.50 (s, 1H),7.5 (s, 1H), 7.28 (m, 1H) 6.95 (m, 2H), 4.21 (s, 2H). ESI-MS: m/z 352.93(M+H)⁺

N-(3-fluoro-4-methoxyphenyl)-2-((6-(trifluoromethyl)-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide(Compound 38)

The reaction mixture of6-(trifluoromethyl)-1H-imidazo[4,5-c]pyridine-2-thiol (15.11 mg, 0.069mmol), 2-chloro-N-(3-fluoro-4-methoxyphenyl)acetamide (15 mg, 0.069mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.029 mL, 0.207mmol) in acetonitrile (1 mL) was heated to 65° C. for 2 hr.Concentration and purification by combiflash (4 g, DCM/MeOH) gaveN-(3-fluoro-4-methoxyphenyl)-2-((6-(trifluoromethyl)-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide(23 mg, 0.057 mmol, 83% yield) 1HNMR (400 MHz, CD₃OD) δ 8.81 (s, 1H),7.89 (s, 1H), 7.51 (m, 1H), 7.20 (m, 1H), 7.02 (t, J=8.8 Hz, 1H), 4.26(s, 2H), 3.33 (s, 3H). ESI-MS: m/z 400.99 (M+H)⁺

2-Chloro-N-(4-(difluoromethoxy)phenyl)acetamide (Compound 39)

To a solution of 4-(difluoromethoxy)aniline (0.078 ml, 0.628 mmol) andTriethylamine (0.131 ml, 0.943 mmol) in DCM at 0° C., was added2-chloroacetyl chloride (0.060 ml, 0.754 mmol). The mixture was stirredat RT for 2 h. Solvent was removed and the crude was purified bycombiflash chromatography (0-30% EtOAc-Hexanes) to afford2-chloro-N-(4-(difluoromethoxy)phenyl)acetamide (110 mg, 0.467 mmol,74.3% yield) as a pale orange solid. ¹H-NMR (400 MHz, CDCl₃) δ ppm 8.24(brs, 1H), 7.55 (dd, 2H, J=2 Hz, J=4 Hz), 7.13 (d, 2H, J=8 Hz), 6.48 (t,1H, J=71.5 Hz), 4.20 (s, 2H); ESI-MS: m/z 235.86 (M+H)⁺.

2-((6-Chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-(difluoromethoxy)phenyl)acetamide(Compound 40)

To a mixture of 2-chloro-N-(4-(difluoromethoxy)phenyl)acetamide (15 mg,0.064 mmol), 6-chloro-3H-imidazo[4,5-c]pyridine-2-thiol (13.00 mg, 0.070mmol) and sodium iodide (9.55 mg, 0.064 mmol) in MeCN was addedtriethylamine (0.013 ml, 0.095 mmol) and the mixture was stirred at 65°C. for 2 h. Solvents removed and the crude was purified by combiflashSiO₂ chromatography (0-100% EtOAc-hexanes) to give2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-(difluoromethoxy)phenyl)acetamide(21 mg, 0.055 mmol, 86% yield) as a white solid. ¹H-NMR (400 MHz,CD₃OD-CDCl₃) δ ppm 8.48 (s, 1H), 7.52 (d, 2H, J=8 Hz), 7.42 (s, 1H),7.05 (d, 1H, J=8 Hz), 6.53 (t, 1H, J=76 Hz), 4.12 (s, 2H); ESI-MS: m/z384.97 (M+H)⁺.

2-Chloro-N-(3-hydroxy-4-methoxyphenyl)propanamide (Compound 41)

A solution of 5-amino-2-methoxyphenol (100 mg, 0.719 mmol) and potassiumcarbonate (109 mg, 0.791 mmol) in acetone/water was cooled and added2-chloropropanoyl chloride (0.084 ml, 0.862 mmol). The mixture wasstirred at −10° C. for 1 h. The reaction was diluted with EtOAc, washedwith 0.5 M HCl, water, brine and dried over anhydrous Na₂SO₄. Solventwas removed and the crude brown solid,2-chloro-N-(3-hydroxy-4-methoxyphenyl)propanamide (130 mg, 0.566 mmol,79% yield), was used in next without purification. 1H-NMR (400 MHz,CDCl₃) δ ppm 7.09 (d, 1H, J=2.4 Hz), 6.96 (dd, 1H, J=2.4 Hz and J=8.8Hz), 6.77 (d, 1H, J=8.8 Hz), 4.47 (q, 1H, J=6.8 Hz), 3.81 (s, 3H), 1.68(d, 3H, J=6.8 Hz); ESI-MS: m/z 229.89 (M+H)⁺.

N-(3-Hydroxy-4-methoxyphenyl)-2-((6-(trifluoromethyl)-3H-imidazo[4,5-c]pyridin-2-yl)thio)propanamide(Compound 42)

A mixture of 2-chloro-N-(3-hydroxy-4-methoxyphenyl)propanamide (12 mg,0.052 mmol), 6-(trifluoromethyl)-3H-imidazo[4,5-c]pyridine-2-thiol(13.74 mg, 0.063 mmol), sodium iodide (3.92 mg, 0.026 mmol) andtriethylamine (10.92 μl, 0.078 mmol) in acetonitrile (1 ml) was stirredat 65° C. for 4 h. Solvent was removed and the crude was purified bycombiflash chromatography (0-5% MeOH-DCM) to obtainN-(3-hydroxy-4-methoxyphenyl)-2-((6-(trifluoromethyl)-3H-imidazo[4,5-c]pyridin-2-yl)thio)propanamide(10 mg, 0.024 mmol, 46.4% yield) as an orange solid. (400 MHz, CD₃OD) δppm 8.84 (s, 1H), 7.91 (s, 1H), 7.12 (d, 1H, J=2.4 Hz), 6.94 (dd, 1H,J=2.4 Hz, J=8.8 Hz), 6.84 (d, 1H, J=8.4 Hz), 4.78 (q, 1H, J=7.2), 3.81(s, 3H), 1.72 (d, 3H, J=7.2 Hz); ESI-MS: m/z 413.02 (M+H)⁺.

2-Chloro-N-(3,5-difluoro-4-methoxyphenyl)acetamide (Compound 43)

A solution of 3,5-difluoro-4-methoxyaniline (100 mg, 0.628 mmol) andtriethylamine (131 μl, 0.943 mmol) in DCM was coiled and added2-chloroacetyl chloride (60.0 μl, 0.754 mmol). The reaction mixture wasstirred at room temperature for 2 h. Reaction was diluted with DCM,washed with 0.5 M HCl, water, brine, dried over Na₂SO₄ and evaporated.The crude brown solid,2-chloro-N-(3,5-difluoro-4-methoxyphenyl)acetamide (120 mg, 0.509 mmol,81% yield) was used for the next step without purification. ¹H-NMR (400MHz, CDCl₃) δ ppm 8.15 (s, 1H), 7.21 (d, 1H, J=8 Hz), 4.18 (s, 1H), 3.97(s, 3H); ESI-MS: m/z 235.82 (M+H)⁺.

N-(3,5-Difluoro-4-methoxyphenyl)-2-((6-(trifluoromethyl)-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide(Compound 44)

A mixture of 2-chloro-N-(3,5-difluoro-4-methoxyphenyl)acetamide (14.19mg, 0.060 mmol), 6-(trifluoromethyl)-3H-imidazo[4,5-c]pyridine-2-thiol(12 mg, 0.055 mmol), sodium iodide (4.10 mg, 0.027 mmol) andtriethylamine (0.011 ml, 0.082 mmol) in MeCN was stirred at 65° C. for 1h. The crude was purified by combiflash chromatography (0-100%EtOAc-hexanes) to giveN-(3,5-difluoro-4-methoxyphenyl)-2-((6-(trifluoromethyl)-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide(20 mg, 0.048 mmol, 87% yield) as a white solid. ¹H-NMR (400 MHz, CD₃OD)δ ppm 8.81 (s, 1H), 7.89 (s, 1H), 7.27 (d, 2H, J=8 Hz), 4.27 (s, 2H),3.89 (s, 3H); ESI-MS: m/z 419.00 (M+H)⁺.

N-(4-fluoro-3-hydroxyphenyl)-2-((6-(trifluoromethyl)-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide(Compound 45)

The reaction mixture of6-(trifluoromethyl)-1H-imidazo[4,5-c]pyridine-2-thiol (12 mg, 0.055mmol), 2-chloro-N-(4-fluoro-3-hydroxyphenyl)acetamide (12.26 mg, 0.060mmol), sodium iodide (8.21 mg, 0.055 mmol), triethylamine (0.023 mL,0.164 mmol) in acetonitrile (1 mL) was heated to 65° C. for 2 hr.Concentration and purification by combiflash (4 g, DCM/MeOH) gaveN-(4-fluoro-3-hydroxyphenyl)-2-((6-(trifluoromethyl)-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide(15 mg, 0.039 mmol, 70.9% yield). ¹HNMR (400 MHz, CD₃OD) δ 8.82 (s, 1H),7.90 (s, 1H), 7.28 (dd, J₁=2.4 Hz, J₂=8 Hz, 1H), 6.94 (m, 2H), 4.25 (s,2H), ESI-MS: m/z 387.03 (M+H)⁺

2-Chloro-3-methyl-5-nitropyridin-4-amine (Compound 46)

To the solution of 2-chloro-3-methylpyridin-4-amine (250 mg, 1.753 mmol)in sulfuric acid (2.63 mL, 49.3 mmol) was added potassium nitroperoxousacid (355 mg, 3.51 mmol) at 0° C. The mixture was warmed to roomtemperature and keep stirred overnight. the mixture was poured into icewater and extracted with EtOAc. The organic layer was dried againstMgSO₄ and concentrated. Purification on combiflash (12 g, hexane/EtOAc)gave 2-chloro-3-methyl-5-nitropyridin-4-amine (157 mg, 0.837 mmol, 47.7%yield) ¹HNMR (400 MHz, CDCl₃), δ 8.96 (s, 1H), 2.32 (s, 3H) ESI-MS: m/z187.84 (M+H)⁺

6-Chloro-5-methylpyridine-3,4-diamine (Compound 47)

The reaction mixture of 2-chloro-3-methyl-5-nitropyridin-4-amine (10 mg,0.053 mmol), iron (14.89 mg, 0.267 mmol), NH₄Cl (14.26 mg, 0.267 mmol)in water (0.5 mL) and ethanol (0.5 mL) was heated to 70° C. for 1.5 hr.Purification by prep-TLC (hexane/EtOAc) gave6-chloro-5-methylpyridine-3,4-diamine (8 mg, 0.051 mmol, 95% yield).¹HNMR (400 MHz, CDCl₃) δ 7.644 (s, 1H), 2.22 (s, 3H) ESI-MS: m/z 157.83(M+H)⁺

6-Chloro-7-methyl-3H-imidazo[4,5-c]pyridine-2-thiol (Compound 48)

The reaction mixture of 6-chloro-5-methylpyridine-3,4-diamine (8 mg,0.051 mmol), di(1H-imidazol-1-yl)methanethione (9.05 mg, 0.051 mmol) intetrahydrofuran (0.5 mL, 0.051 mmol). Overnight. prep-TLC gave6-chloro-7-methyl-3H-imidazo[4,5-c]pyridine-2-thiol (6 mg, 0.030 mmol,59.2% yield) ¹HNMR (400 MHz, CD₃OD) δ 7.996 (s, 1H). 2.44 (s, 3H)ESI-MS: m/z 199.79 (M+H)⁺

2-((6-Chloro-7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-hydroxy-4-methoxyphenyl)acetamide(Compound 49)

The reaction mixture of6-chloro-7-methyl-1H-imidazo[4,5-c]pyridine-2-thiol (6 mg, 0.030 mmol),2-chloro-N-(3-hydroxy-4-methoxyphenyl)acetamide (6.48 mg, 0.030 mmol),sodium iodide (1 mg, 6.67 μmol), triethylamine (0.013 mL, 0.090 mmol) inAcetonitrile (1 mL) was heated to 65° C. for 2 hr. Concentration andpurification by prep-TLC (10%, DCM/MeOH) gave2-((6-chloro-7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-hydroxy-4-methoxyphenyl)acetamide(7 mg, 0.018 mmol, 61.5% yield) ¹HNMR (400 MHz, CD₃OD) δ 8.36 (s, 1H),7.11 (d, J=2 Hz, 1H), 6.95 (dd, J₁=2 Hz, J₂=8.8 Hz, 1H), 6.84 (8.8 Hz,1H), 4.19 (s, 2H), 3.011 (s, 3H), 2.54 (s, 3H). ESI-MS: m/z 379.00(M+H)⁺

2-((6-Chloro-7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-fluoro-4-methoxyphenyl)acetamide(Compound 50)

The reaction mixture of6-chloro-7-methyl-1H-imidazo[4,5-c]pyridine-2-thiol (10 mg, 0.050 mmol),2-chloro-N-(3-fluoro-4-methoxyphenyl)acetamide (10.90 mg, 0.050 mmol),sodium iodide (1 mg, 6.67 μmol), triethylamine (0.021 mL, 0.150 mmol) inacetonitrile (1 mL) was heated at 65° C. for 2 hr. Concentration andpurification on prep-TLC (15%, MeOH/DCM) gave2-((6-chloro-7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-fluoro-4-methoxyphenyl)acetamide(3.1 mg, 8.14 μmol, 16.25% yield) ¹HNMR (400 MHz, CD₃OD) δ 8.36 (s, 1H),7.50 (d, J=13.4 Hz, 1H), 7.18 (d, J=8.4 Hz, 1H), 7.03 (t, J=9.2 Hz, 1H),4.21 (s, 2H), 3.84 (s, 3H) 2.53 (s, 3H), ESI-MS: m/z 381.05 (M+H)⁺

2-((6-Chloro-7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-hydroxyphenyl)acetamide(Compound 51)

The reaction mixture of6-chloro-7-methyl-1H-imidazo[4,5-c]pyridine-2-thiol (10 mg, 0.050 mmol),2-chloro-N-(4-fluoro-3-hydroxyphenyl)acetamide (10.20 mg, 0.050 mmol),sodium iodide (1 mg, 6.67 mmol), triethylamine (0.021 mL, 0.150 mmol) inAcetonitrile (1 mL) was heated at 65° C. for 2 hr. Concentration andpurification by prep-TLC (15% MeOH/DCM) gave2-((6-chloro-7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-hydroxyphenyl)acetamide(14.5 mg, 0.040 mmol, 79% yield) ¹HNMR (400 MHz, CD₃OD), 8.36 (s, 1H),7.28 (m, 1H), 6.97 (m, 2H), 4.20 (s, 2H), 2.54 (s, 3H), ESI-MS: m/z367.00 (M+H)⁺

2-((6-Chloro-7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3,4-dimethoxyphenyl)acetamide(Compound 52)

The reaction mixture of6-chloro-7-methyl-1H-imidazo[4,5-c]pyridine-2-thiol (10 mg, 0.050 mmol),2-chloro-N-(3,4-dimethoxyphenyl)acetamide (11.50 mg, 0.050 mmol), sodiumiodide (1 mg, 6.67 μmol), triethylamine (0.021 mL, 0.150 mmol) inacetonitrile (1 mL) was heated at 65° C. for 2 hr. Filtration gave2-((6-chloro-7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3,4-dimethoxyphenyl)acetamide(7.4 mg, 0.019 mmol, 37.6% yield) ¹HNMR (400 MHz, CD₃OD) δ 8.37 (s, 1H),7.30 (d, J=2.4 Hz, 1H), 7.01 (m, 6.88 (d, J=8.8 Hz, 1H), 4.22 (s, 2H),3.79 (s, 6H), 2.54 (s, 3H) ESI-MS: m/z 393.05 (M+H)⁺

2-Chloro-N-(5,6-dimethoxybenzo[d]thiazol-2-yl)acetamide (Compound 53)

To the reaction mixture of 5,6-dimethoxybenzo[d]thiazol-2-amine (50 mg,0.238 mmol), TEA (0.100 mL, 0.713 mmol) in DCM (1 mL) was added2-chloroacetyl chloride (0.023 mL, 0.285 mmol) at room temperature.Overnight, concentration and purification on combiflash (4 g, MeOH/DCM)gave 2-chloro-N-(5,6-dimethoxybenzo[d]thiazol-2-yl)acetamide (15 mg,0.052 mmol, 22.00% yield). 1HNMR (400 MHz, CDCl₃), 9.73 (s, 1H), 7.26(s, 1H), 7.25 (s, 1H), 4.30 (s, 2H), 3.969s, 6H) ESI-MS: m/z 286.94(M+H)⁺

2-((6-Chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(5,6-dimethoxybenzo[d]thiazol-2-yl)acetamide(Compound 54)

The reaction mixture of 6-chloro-3H-imidazo[4,5-c]pyridine-2-thiol (6.47mg, 0.035 mmol), 2-chloro-N-(5,6-dimethoxybenzo[d]thiazol-2-yl)acetamide(10 mg, 0.035 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine(0.015 mL, 0.105 mmol) in CH₃CN (1 mL) was heated at 65° C. for 2 hr.Concentration and combiflash (4 g, DCM/MeOH) gave2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(5,6-dimethoxybenzo[d]thiazol-2-yl)acetamide(8 mg, 0.018 mmol, 52.6% yield) ¹HNMR (400 MHz, CD₃OD) δ 8.12 (s, H),7.41 (s, 1H), 7.33 (s, 2H), 4.38 (s, 2H), 3.89 (s, 3H), 3.88 (s, 3H),ESI-MS: m/z 436.00 (M+H)⁺

Methyl (2-(trifluoromethoxy)phenyl)carbonate (Compound 55)

To a cooled solution of 2-(trifluoromethoxy)phenol (0.51 ml, 3.93 mmol)and triethylamine (1.1 ml, 7.86 mmol) in DCM was slowly added methylchloroformate (0.36 ml, 4.72 mmol) and the reaction was stirred at 25°C. for 2 h. Reaction diluted with DCM and washed with water, brine,dried over anhydrous Na₂SO₄ and evaporated. The crude cloudy oil (750mg, 81%) was used in next step without further purification. ¹H-NMR (400MHz, CDCl₃) δ ppm 7.30 (m, 4H), 3.93 (s, 3H); ¹⁹F-NMR (376.5 MHz, CDCl₃)δ ppm −58.10 (s, 3F).

Methyl (5-nitro-2-(trifluoromethoxy)phenyl)carbonate (Compound 57)

A suspension of methyl (2-(trifluoromethoxy)phenyl)carbonate (750 mg,3.18 mmol) in sulfuric acid (2.0 ml, 37.5 mmol) was cooled and solidpotassium nitrate (250 mg, 2.473 mmol) was added portion wise. Themixture was stirred at room temperature for 2 h. Reaction mixture waspoured into ice water and extracted with DCM. The combined extracts werewashed with water, brine and dried over Na₂SO₄. Solvent was removed andthe crude was purified by combiflash SiO₂ chromatography (0-5%EtOAc-hexanes) to afford methyl(4-nitro-2-(trifluoromethoxy)phenyl)carbonate (250 mg, 28%) and methyl(5-nitro-2-(trifluoromethoxy)phenyl)carbonate (553 mg, 62%) as tansolid.

Methyl (5-nitro-2-(trifluoromethoxy)phenyl)carbonate (Less Polar)

¹H-NMR (400 MHz, CDCl₃) δ ppm 8.23 (m, 1H), 8.21 (dd, 1H, J=2.4, 8.8Hz), 7.52 (dd, 1H, J=1.2, 9.2 Hz), 3.98 (s, 3H); ¹⁹F-NMR (376.5 MHz,CDCl₃) δ ppm −57.77 (s, 3F)

Methyl (4-nitro-2-(trifluoromethoxy)phenyl)carbonate (Compound 56)(Polar)

¹H-NMR (400 MHz, CDCl₃) δ ppm 8.25 (m, 2H), 7.52 (dd, 1H, J=4.4, 5.2Hz), 3.97 (s, 3H); ¹⁹F-NMR (376.5 MHz, CDCl₃) δ ppm −58.23 (s, 3F).

5-Amino-2-(trifluoromethoxy)phenyl Methyl Carbonate (Compound 58)

A solution of methyl (5-nitro-2-(trifluoromethoxy)phenyl)carbonate (190mg, 0.676 mmol) and ammonium chloride (108 mg, 2.027 mmol) in EtOH-water(1:1) was added iron (113 mg, 2.027 mmol) and the mixture was stirred at100° C. for 30 min in a microwave reactor. Solvents removed and thecrude was purified by combiflash SiO₂ chromatography (0-30%EtOAc-hexanes) to give 5-amino-2-(trifluoromethoxy)phenyl methylcarbonate (66 mg, 39%) as a brown solid. ¹H-NMR (400 MHz, CDCl₃) δ ppm7.09 (dd, 1H, J=0.8, 8.4 Hz), 6.54 (d, 1H, J=2.8 Hz), 6.51 (dd, 1H,J=2.8, 8.8 Hz), 3.91 (s, 3H); ¹⁹F-NMR (376.5 MHz, CDCl₃) δ ppm −58.82(s, 3F).

5-Amino-2-(trifluoromethoxy)phenol (Compound 59)

A solution of 5-amino-2-(trifluoromethoxy)phenyl methyl carbonate (66mg, 0.263 mmol) and potassium carbonate (73 mg, 0.526 mmol) inMeOH-water (2:1) was stirred at room temperature for 1 h. MeOH wasremoved and the reaction was neutralized with 0.5 M HCl and extractedwith EtOAc. The combined organic layers were washed with brine, driedover Na₂SO₄ and evaporated. The crude brown solid,5-amino-2-(trifluoromethoxy)phenol (40 mg, 79%), was used in next stepwithout purification. 1H-NMR (400 MHz, CD₃OD) δ ppm 6.88 (dd, 1H, J=0.8,8.8 Hz), 6.29 (d, 1H, J=2.8 Hz), 6.17 (dd, 1H, J=2.4, 8.8 Hz); ¹⁹F-NMR(376.5 MHz, CDCl₃) δ ppm −59.92 (s, 3F); ESI-MS: m/z 193.85 (M+H)⁺.

2-Chloro-N-(3-hydroxy-4-(trifluoromethoxy)phenyl)acetamide (Compound 60)

To a cooled solution of 5-amino-2-(trifluoromethoxy)phenol (90 mg, 0.466mmol) and potassium carbonate (77 mg, 0.559 mmol) in acetone-water (3:1)was slowly added 2-chloroacetyl chloride (0.044 ml, 0.559 mmol) andstirred at 0° C. for 2 h. Solvents removed and the crude was purified bycombiflash SiO₂ chromatography (0-50% EtOAc-hexanes) to give2-chloro-N-(3-hydroxy-4-(trifluoro-methoxy)phenyl)acetamide (98 mg, 78%)as a brown solid. ¹H-NMR (400 MHz, CD₃OD) δ ppm 7.40 (d, 1H, J=2.4 Hz),7.10 (d, 1H, J=8.8 Hz), 6.95 (dd, 1H, J=2.0, 8.8 Hz, 4.12 (s, 2H);¹⁹F-NMR (376.5 MHz, CDCl₃) δ ppm −59.92 (s, 3F); ESI-MS: m/z 269.92(M+H)⁺.

N-(3-Hydroxy-4-(trifluoromethoxy)phenyl)-2-((6-(trifluoromethyl)-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide(Compound 61)

A mixture of 2-chloro-N-(3-hydroxy-4-(trifluoromethoxy)phenyl)acetamide(12 mg, 0.045 mmol),6-(trifluoromethyl)-3H-imidazo[4,5-c]pyridine-2-thiol (11.71 mg, 0.053mmol), triethylamine (9.31 μl, 0.067 mmol) and sodium iodide (3.34 mg,0.022 mmol) in MeCN was stirred at 65° C. for 1 h. The crude purified bycombiflash SiO₂ chromatography (0-5% MeOH-DCM) to giveN-(3-hydroxy-4-(trifluoromethoxy)phenyl)-2-((6-(trifluoromethyl)-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide(11 mg, 55%) as off white solid. ¹H-NMR (400 MHz, CD₃OD) δ ppm 8.79 (s,1H), 7.87 (s, 1H), 7.39 (d, 1H, J=2.4 Hz), 7.12 (dd, 1H, J=1.2, 8.8 Hz),6.97 (dd, 1H, J=2.8, 8.8 Hz), 4.23 (s, 2H); ¹⁹F-NMR (376.5 MHz, CDCl₃) δppm −60.12 (s, 3F), −67.41 (s, 3F); ESI-MS: m/z 453.01 (M+H)⁺.

N-(Benzo[d]thiazol-2-yl)-2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide(Compound 62)

To a mixture of 6-chloro-3H-imidazo[4,5-c]pyridine-2-thiol (10 mg, 0.054mmol), N-(benzo[d]thiazol-2-yl)-2-chloroacetamide (14.7 mg, 0.065 mmol)and sodium iodide (4.0 mg, 0.027 mmol) in MeCN was added triethylamine(11 μL, 0.081 mmol) and the reaction was stirred at 65° C. for 1 h.Solvents removed and the crude was purified by combiflash SiO₂chromatography (0-5% MeOH-DCM) to giveN-(benzo[d]thiazol-2-yl)-2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide(16 mg, 79%) as a white solid. ¹H-NMR (400 MHz, DMSO-d₆) δ ppm 8.50 (s,1H), 7.95 (d, 1H, J=8 Hz), 7.75 (d, 1H, J=8 Hz), 7.51 (s, 1H), 7.43 (t,1H, J=7.2 Hz), 7.29 (t, 1H, J=7.2 Hz), 4.46 (s, 2H); ESI-MS: m/z 375.98(M)⁺, 377.99 (M+2H)⁺.

2-Bromo-5-nitropyridin-4-amine (Compound 63)

To a solution of 2,4-dibromo-5-nitropyridine (200 mg, 0.709 mmol) andtriethylamine (0.2 ml, 1.419 mmol) in THF 7 M ammonia (0.2 ml, 1.419mmol) was added and stirred at room temperature for 24 h. Solvents wereremoved and the crude was purified by combiflash SiO₂ chromatography(0-50% EtOAc-hexanes) to give 2-bromo-5-nitropyridin-4-amine (135 mg,87%) as a tan solid. 1H-NMR (400 MHz, CD₃OD) δ ppm 8.82 (s, 1H), 7.11(s, 1H); m/z 219.69 (M+H)⁺.

4-Amino-5-nitropicolinonitrile (Compound 64)

A solution of 2-bromo-5-nitropyridin-4-amine (135 mg, 0.619 mmol) andcopper cyanide (67 mg, 0.743 mmol) in DMA was heated to 200° C. for 1 husing a microwave reactor. The reaction mixture was partitioned betweenwater and EtOAc and tilted over celite. The aqueous layer was extractedwith EtOAc. The combined organic layers were washed with water, brine,dried over Na₂SO₄ and concentrated under reduced pressure. The crude waspurified by combiflash SiO₂ chromatography using (0-50% EtOAc-hexanes)to give 4-amino-5-nitropicolinonitrile (70 mg, 69%) as a pale brownsolid. ¹H-NMR (400 MHz, CD₃OD) δ ppm 9.07 (s, 1H), 7.37 (s, 1H); ESI-MS:m/z 164.77 (M+H)⁺.

4,5-Diaminopicolinonitrile (Compound 65)

A slurry of 4-amino-5-nitropicolinonitrile (70 mg, 0.427 mmol) and iron(72 mg, 1.280 mmol) in EtOH was added ammonium chloride (68 mg, 1.280mmol) in water and the mixture was heated to 65° C. for 1 h. Thereaction mixture was filtered through celite and the solvents wereremoved. The crude was purified by combiflash SiO₂ chromatography (0-5%MeOH-DCM) to give 4,5-diaminopicolinonitrile (32 mg, 56%) as a palebrown solid. ¹H-NMR (400 MHz, CD₃OD) δ ppm 7.72 (s, 1H), 6.92 (s, 1H);ESI-MS: m/z 134.85 (M+H)⁺.

2-Mercapto-3H-imidazo[4,5-c]pyridine-6-carbonitrile (Compound 66)

A solution of 4,5-diaminopicolinonitrile (32 mg, 0.239 mmol),di(1H-imidazol-1-yl)methanethione (51 mg, 0.286 mmol) andN,N-dimethylpyridin-4-amine (29 mg, 0,239 mmol) in MeCN was stirred atroom temperature for 16 h. MeCN was removed and the crude was purifiedby combiflash SiO₂ chromatography (0-5% MeOH-DCM) to give2-mercapto-3H-imidazo[4,5-c]pyridine-6-carbonitrile (30 mg, 71%) as tansolid. ¹H-NMR (400 MHz, CD₃OD) δ ppm 8.43 (s, 1H), 7.64 (s, 1H); ESI-MS:m/z 176.91 (M+H)⁺.

2-((6-Cyano-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-hydroxy-4-methoxyphenyl)acetamide(Compound 67)

A mixture of 2-chloro-N-(3-hydroxy-4-methoxyphenyl)acetamide (3.3 mg,0.015 mmol), 2-mercapto-3H-imidazo[4,5-c]pyridine-6-carbonitrile (3 mg,0.017 mmol) and sodium iodide (1.2 mg, 7.74 μmol) in MeCN was addedtriethylamine (2.6 μL, 0.019 mmol) and the solution was stirred for 1 hat 65° C. Solvent was removed and the crude was purified by combiflashSiO₂ chromatography (0-5% MeOH-DCM) to give2-((6-cyano-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-hydroxy-4-methoxyphenyl)acetamide(5 mg, 91%) as an off-white solid. ¹H-NMR (400 MHz, CD₃OD-CDCl₃) δ ppm8.77 (s, 1H), 7.93 (s, 1H), 7.10 (d, 1H, J=2.8 Hz), 6.94 (dd, 1H, J=2.8,8.8 Hz), 6.79 (d, 1H, J=8.8 Hz), 4.15 (s, 2H), 3.80 (s, 3H); ESI-MS: m/z375.98 (M)⁺, 356.04 (M+2H)⁺.

2-((6-Cyano-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3,4-dimethoxyphenyl)acetamide(Compound 68)

A mixture of 2-chloro-N-(3,4-dimethoxyphenyl)acetamide (14 mg, 0.062mmol), 2-mercapto-3H-imidazo[4,5-c]pyridine-6-carbonitrile (10 mg, 0.057mmol) and sodium iodide (4.3 mg, 0.028 mmol) in MeCN was addedtriethylamine (12 μL, 0.085 mmol) and stirred at 65° C. for 1 h. MeCNwas removed and the crude was purified by combiflash SiO₂ chromatography(0-5% MeOH-DCM) to afford2-((6-cyano-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3,4-dimethoxyphenyl)acetamide(15 mg, 72%) as a white solid. ¹H-NMR (400 MHz, CD₃OD-CDCl₃) δ ppm 8.80(s, 1H), 7.98 (s, 1H), 7.33 (d, 1H, J=2.4 Hz), 7.01 (dd, 1H, J=2.4, 8.8Hz), 6.87 (d, 1H, J=8.8 Hz), 4.24 (s, 2H), 3.81 (s, 3H), 3.30 (s, 3H);ESI-MS: m/z 370.05 (M+H)⁺.

2-((6-Cyano-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-hydroxyphenyl)acetamide(Compound 69)

A mixture of 2-chloro-N-(4-fluoro-3-hydroxyphenyl)acetamide (13 mg,0.062 mmol), 2-mercapto-3H-imidazo[4,5-c]pyridine-6-carbonitrile (10 mg,0.057 mmol) and sodium iodide (4.3 mg, 0.028 mmol) in MeCN was addedtriethylamine (12 μL, 0.085 mmol) and stirred at 65° C. for 1 h. MeCNwas removed and the crude was purified by combiflash SiO₂ chromatography(0-5% MeOH-DCM) to afford2-((6-cyano-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-hydroxyphenyl)acetamide(11 mg, 57%) as a white solid. ¹H-NMR (400 MHz, CD₃OD-CDCl₃) δ ppm 8.79(s, 1H), 7.95 (s, 1H), 7.25 (dd, 1H, J=2.4, 7.6 Hz), 6.95 (d, 1H, J=8.8Hz), 6.91 (m, 1H), 4.19 (s, 2H); ESI-MS: m/z 344.01 (M+H)⁺.

2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)sulfonyl)-N-(3-fluoro-4-methoxyphenyl)acetamide(Compound 27)

To the solution of2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-fluoro-4-methoxyphenyl)acetamide(5 mg, 0.014 mmol) in methanol (1 mL, 0.014 mmol) was added Oxone (20.95mg, 0.068 mmol). Overnight, the product was purified on Prep-TLC(MeOH/DCM) gave2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)sulfonyl)-N-(3-fluoro-4-methoxyphenyl)acetamide(2.1 mg, 5.27 μmol, 38.6% yield). ¹HNMR (400 MHz, CD₃OD) δ 8.72 (s, 1H),7.62 (s, 1H), 7.40 (M, 1H), 7.13 (d, J=7.6 Hz, 1H), 7.00 (m, 1H), 3.83(s, 3H) ESI-MS: m/z 399.0 (M+H)⁺

2-Chloro-N-(6-methoxypyridin-3-yl)acetamide (Compound 71)

To the solution of 6-methoxypyridin-3-amine (53 mg, 0.427 mmol), TEA(0.179 mL, 1.281 mmol) in DCM (2 mL) was added 2-chloroacetyl chloride(0.037 mL, 0.470 mmol) at room temperature. After 1 hr, Concentrationand purification on combiflash (4 g, EtOAc/Hexane) gave2-chloro-N-(6-methoxypyridin-3-yl)acetamide (51 mg, 0.254 mmol, 59.5%yield). ¹HNMR (400 MHz, CDCl₃) δ 8.30 (d, J=2.4 Hz, 1H), 7.88 (m, 1H),6.79 (d, J=8.8 Hz, 1H), 4.18 (s, 2H). ESI-MS: m/z 200.98 (M+H)⁺

2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(6-methoxypyridin-3-yl)acetamide(Compound 72)

The reaction mixture of 6-chloro-3H-imidazo[4,5-c]pyridine-2-thiol (9.25mg, 0.050 mmol), 2-chloro-N-(6-methoxypyridin-3-yl)acetamide (10 mg,0.050 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.021 mL,0.150 mmol) in CH3CN (1 mL) was heated at 65° C. for 2 hr. Filtrationgave2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(6-methoxypyridin-3-yl)acetamide(12 mg, 0.034 mmol, 68.8% yield) ¹HNMR (400 MHz, CD₃OD) δ 8.63 (s, 1H),8.43 (d, J=2.4 Hz, 1H), 8.01 (m 1H), 7.62 (s, 1H), 6.90 (d, J=8.8 Hz,1H), 4.38 (s, 2H), 4.00 (s, 3H)

ESI-MS: m/z 349.95 (M+H)⁺

2-Bromo-3-methyl-5-nitropyridin-4-amine (Compound 73)

A mixture of 2-bromo-3-methylpyridin-4-amine (100 mg, 0.535 mmol) insulfuric acid (849 μl, 16.04 mmol) was cooled to 0° C., and addedpotassium nitrate (162 mg, 1.604 mmol). The mixture was stirred at 0° C.for additional 1 h and at room temperature for 16 h. The reactionmixture was poured into ice water, basified with sat. NaHCO₃ andextracted with EtOAc. The combined EtOAc layers were washed with water,brine, dried over Na₂SO₄ and evaporated. The crude was purified bycombiflash SiO₂ chromatography (0-20% EtOAc-hexanes) to give2-bromo-3-methyl-5-nitropyridin-4-amine (54 mg, 44%) as a yellow solid.¹H-NMR (400 MHz, CDCl₃—CD₃OD) δ ppm 8.69 (s, 1H), 2.22 (s, 3H); ESI-MS:m/z 231.86 (M⁺) 233.85 (M+2H)⁺.

4-Amino-3-methyl-5-nitropicolinonitrile (Compound 74)

A solution of 2-bromo-3-methyl-5-nitropyridin-4-amine (52 mg, 0.224mmol) and cyanocopper (24.09 mg, 0.269 mmol) in DMA was heated to 200°C. in a microwave reactor for 1 h. The reaction mixture was poured intowater and extracted with EtOAc. The combined organic layers were washedwith water, brine, dried over Na₂SO₄ and evaporated. The crude waspurified by combiflash SiO₂ chromatography (0-30% EtOAc-hexanes) to give4-amino-3-methyl-5-nitropicolinonitrile (30 mg, 75%) as a pale yellowsolid. ¹H-NMR (400 MHz, CD₃OD) δ ppm 8.99 (s, 1H), 2.44 (s, 3H); ESI-MS:m/z 178.93 (M+H)⁺.

4,5-Diamino-3-methylpicolinonitrile (Compound 75)

A solution of 4-amino-3-methyl-5-nitropicolinonitrile (30 mg, 0.168mmol) in EtOH was added iron (9.40 mg, 0.168 mmol) followed by asolution of ammonium chloride (9.01 mg, 0.168 mmol) in water and stirredat 65° C. for 1 h. The reaction mixture was filtered through celite andevaporated. The crude was purified by combiflash SiO₂ chromatography(0-5% MeOH-DCM) to give 4,5-diamino-3-methylpicolinonitrile (15 mg, 60%)as a brown solid. ¹H-NMR (400 MHz, CD₃OD) δ ppm 7.77 (s, 1H), 2.29 (s,3H). ESI-MS: m/z 148.97 (M+H)⁺

2-Mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (Compound76)

A mixture of 4,5-diamino-3-methylpicolinonitrile (15 mg, 0.101 mmol),di(1H-imidazol-1-yl)methanethione (27.1 mg, 0.152 mmol) andN,N-dimethylpyridin-4-amine (14.84 mg, 0.121 mmol) in MeCN was stirredat room temperature for 16 h. Solvent was removed and the crude waspurified by combiflash SiO₂ chromatography (0-5% MeOH-DCM) to give2-mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (17 mg,88%) as a pale brown solid. ¹H-NMR (400 MHz, CD₃OD) δ ppm 8.31 (s, 1H),2.59 (s, 3H); ESI-MS: m/z 190.93 (M+H)⁺.

2-((6-Cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3,4-dimethoxyphenyl)acetamide(Compound 77)

A mixture of2-mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (3 mg,0.016 mmol), 2-chloro-N-(3,4-dimethoxyphenyl)acetamide (4.35 mg, 0.019mmol) and sodium iodide (1.182 mg, 7.89 μmol) in MeCN was addedtriethylamine (3.30 μl, 0.024 mmol) and heated to 65° C. for 1 h.Solvent was removed and the crude was purified by combiflash SiO₂chromatography (0-10% MeOH-DCM) to give2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3,4-dimethoxyphenyl)acetamide(4 mg, 66%) as a white solid. ¹H-NMR (400 MHz, CDCl₃—CD₃OD) δ ppm 8.64(s, 1H), 7.32 (s, 1H), 6.98 (d, 1H, J=7.6 Hz), 6.84 (dd, 1H, J=3.2, 8.8Hz), 4.20 (s, 2H), 3.82 (s, 3H), 3.81 (s, 3H), 2.71 (s, 3H); ESI-MS: m/z384.06 (M+H)⁺.

2-((6-Cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-hydroxy-4-methoxyphenyl)acetamide(Compound 78)

A mixture of2-mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (7 mg,0.037 mmol), 2-chloro-N-(3-hydroxy-4-methoxyphenyl)acetamide (8.73 mg,0.040 mmol), sodium iodide (2.76 mg, 0.018 mmol) in MeCN was addedtriethylamine (7.69 μl, 0.055 mmol) and heated to 65° C. for 1 h.Solvent was removed and the crude was purified by combiflash SiO₂chromatography (0-10% MeOH-DCM) to give2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-hydroxy-4-methoxyphenyl)acetamide(7 mg, 52%) as a white solid. ¹H-NMR (400 MHz, CDCl₃-CD₃OD) δ ppm 8.64(s, 1H), 7.10 (d, 1H, J=2.4 Hz), 6.94 (dd, 1H, J=2.4, 8.8 Hz), 6.84 (d,1H, J=8.8 Hz), 4.18 (s, 2H), 3.82 (s, 3H), 3.81 (s, 3H), 2.71 (s, 3H);ESI-MS: m/z 370.05 (M+H)⁺.

2-((6-Cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-hydroxyphenyl)acetamide(Compound 79)

A mixture of2-mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (11.21 mg,0.059 mmol), 2-chloro-N-(4-fluoro-3-hydroxyphenyl)acetamide (10 mg,0.049 mmol) and sodium iodide (3.68 mg, 0.025 mmol) in MeCN was addedtriethylamine (10.27 μl, 0.074 mmol) and stirred at 65° C. for 1 h. MeCNwas removed and the crude was purified by combiflash SiO₂ chromatography(0-5% MeOH-DCM) to afford2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-hydroxyphenyl)acetamide(10 mg, 57%) as a white solid. ¹H-NMR (400 MHz, CDCl₃-CD₃OD) δ ppm 8.22(s, 1H), 6.82 (dd, 1H, J=2.4, 8.0 Hz), 6.49 (m, 2H), 3.76 (s, 2H), 2.29(s, 3H); ESI-MS: m/z 358.03 (M+H)⁺.

2-Chloro-N-(4-methoxy-3-nitrophenyl)acetamide (Compound 80)

A solution of 4-methoxy-3-nitroaniline (100 mg, 0.595 mmol) in acetonewas mixed with a solution of potassium carbonate (123 mg, 0.892 mmol) inwater and cooled before the addition of 2-chloroacetyl chloride (0.061ml, 0.773 mmol). The reaction mixture was stirred at 0° C. for 1 h. Thereaction mixture was diluted with EtOAc, washed with 1 M HCl, sat.NaHCO₃, water, brine and dried over Na₂SO₄. The solvent was removed andthe crude, 2-chloro-N-(4-methoxy-3-nitrophenyl)acetamide (120 mg, 82%)as a yellow solid, was used in the next step without furtherpurification. ¹H-NMR (400 MHz, CDCl₃) δ ppm 8.25 (brs, 1H), 8.06 (d, 1H,J=2.4 Hz), 7.80 (dd, 1H, J=2.4, 8.8 Hz), 7.09 (d, 1H, J=8.8 Hz), 4.21(s, 2H), 3.96 (s, 3H), 3.81 (s, 3H); ESI-MS: m/z 244.98 (M+H)⁺, 266.94(M+Na)⁺.

N-(3-Amino-4-methoxyphenyl)-2-chloroacetamide (Compound 81)

A mixture of 2-chloro-N-(4-methoxy-3-nitrophenyl)acetamide (50 mg, 0.204mmol) and iron (34.2 mg, 0.613 mmol) in EtOH as added a solution ofammonium chloride (32.8 mg, 0.613 mmol) and the mixture was stirred at65° C. for 1 h. The reaction was filtered through celite and thesolvents were removed. The crude brown solid,N-(3-amino-4-methoxyphenyl)-2-chloroacetamide (30 mg, 68%), was used inthe next step without further purification. ¹H-NMR (400 MHz,CDCl₃—CD₃OD) δ ppm 7.08 (s, 1H), 6.89 (d, 1H, J=8 Hz), 6.79 (d, 1H,J=8.8 Hz), 4.15 (s, 2H), 3.82 (s, 3H), 3.81 (s, 3H); ESI-MS: m/z 214.99(M+H)⁺.

N-(3-Amino-4-methoxyphenyl)-2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide(Compound 82)

A mixture of N-(3-amino-4-methoxyphenyl)-2-chloroacetamide (15 mg, 0.070mmol), 6-chloro-3H-imidazo[4,5-c]pyridine-2-thiol (10.81 mg, 0.058 mmol)and sodium iodide (4.36 mg, 0.029 mmol) in MeCN was added triethylamine(12.18 μl, 0.087 mmol) and stirred at 65° C. for 1 h. MeCN was removedand the crude was purified by combiflash SiO₂ chromatography (0-5%MeOH-DCM) to affordN-(3-amino-4-methoxyphenyl)-2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide(5.5 mg, 26%) as a white solid. ¹H-NMR (400 MHz, CDCl₃—CD₃OD) δ ppm 8.51(s, 1H), 7.50 (s, 1H), 7.01 (d, 1H, J=2.4 Hz), 6.83 (dd, 1H, J=2.4, 8.8Hz), 6.76 (d, 1H, J=8.8 Hz), 4.19 (s, 2H), 3.82 (s, 3H), 3.81 (s, 3H);ESI-MS: m/z 363.97 (M+H)⁺.

2-((6-Chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-methoxy-3-nitrophenyl)acetamide(Compound 83)

A mixture of 2-chloro-N-(4-methoxy-3-nitrophenyl)acetamide (15.8 mg,0.065 mmol), 6-chloro-3H-imidazo[4,5-c]pyridine-2-thiol (10 mg, 0.054mmol) and sodium iodide (8.07 mg, 0.054 mmol) in MeCN was addedtriethylamine (11 μl, 0.081 mmol) and stirred at 65° C. for 1 hr. MeCNwas removed and the crude was purified by combiflash SiO2 chromatography(0-5% MeOH-DCM) to afford2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-methoxy-3-nitro-phenyl)acetamide(17 mg, 80%) as a white solid. ¹H-NMR (400 MHz, DMSO-d6) δ ppm 10.72 (s,1H), 8.51 (s, 1H), 8.22 (d, 1H, J=2.4 Hz), 7.72 (dd, 1H, J=2.4, 8.8 Hz),7.52 (s, 1H), 7.35 (d, 1H, J=8.8 Hz), 4.32 (s, 2H), 3.87 (s, 3H);ESI-MS: m/z 393.98 (M⁺).

2-Chloro-N-(4,5-dimethoxypyridin-2-yl)acetamide (Compound 84)

To the solution of 4,5-dimethoxypyridin-2-amine (50 mg, 0.324 mmol), TEA(0.113 mL, 0.811 mmol) in DCM (2 mL) was added 2-chloroacetyl chloride(0.028 mL, 0.357 mmol) at room temperature. After 1 hr, concentrationand purification on combiflash (4 g, EtOAc/Hexane) gave2-chloro-N-(4,5-dimethoxypyridin-2-yl)acetamide (19 mg, 0.082 mmol,25.4% yield). ¹HNMR (400 MHz, CDCl₃) δ ppm 8.72 (s, 1H), 7.87 (s, 1H),7.8 (s, 1H), 4.18 (s, 2H), 3.97 (s, 3H), 3.92 (s, 3H) ESI-MS: m/z 230.91(M+H)⁺

2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4,5-dimethoxypyridin-2-yl)acetamide(Compound 85)

The reaction mixture of 6-chloro-1H-imidazo[4,5-c]pyridine-2-thiol (7.24mg, 0.039 mmol), 2-chloro-N-(4,5-dimethoxypyridin-2-yl)acetamide (9 mg,0.039 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.016 mL,0.117 mmol) in CH₃CN (1 mL) was heated to 65° C. for 2 hr. Concentrationand purification on combiflash (4 g, MeOH/DCM) gave2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4,5-dimethoxypyridin-2-yl)acetamide(9 mg, 0.024 mmol, 60.7% yield) 1HNMR (400 MHz, CD₃OD) δ ppm 8.52 (s,1H), 7.82 (s, 1H), 7.78 (s, 1H), 7.51 (s, 4.26 (s, 2H), 3.88 (s, 3H),3.85 (s, 3H). ESI-MS: m/z 379.96 (M+H)⁺

2-Chloro-N-(5,6-dimethoxypyridin-2-yl)acetamide (Compound 86)

To the solution of 5,6-dimethoxypyridin-2-amine (100 mg, 0.649 mmol),TEA (0.226 mL, 1.622 mmol) in DCM (3 mL) was added 2-chloroacetylchloride (0.057 mL, 0.714 mmol) at room temperature. After 1 hr,concentration and purification on combiflash (4 g, EtOAc/Hexane) gave2-chloro-N-(5,6-dimethoxypyridin-2-yl)acetamide (85 mg, 0.369 mmol,56.8% yield). ¹HNMR (400 MHz, CDCl₃) δ ppm 8.75 (s, 1H), 7.95 (d, J=8Hz, 1H), 7.54 (s, 1H), 7.37 (d, J=8 Hz, 1H), 4.46 (s, 2H), 4.25 (s, 3H),4.20 (s, 3H). ESI-MS: m/z 231.12 (M+H)⁺

2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(5,6-dimethoxypyridin-2-yl)acetamide(Compound 87)

The reaction mixture of 6-chloro-1H-imidazo[4,5-c]pyridine-2-thiol (8.05mg, 0.043 mmol), 2-chloro-N-(5,6-dimethoxypyridin-2-yl)acetamide (10 mg,0.043 mmol), sodium iodide (1 mg, 6.67 μmol) and triethylamine (0.018mL, 0.130 mmol) in CH₃CN (1 mL) was heated to 65° C. overnight. Two newsports was purified by pre-TLC (MeOH/DCM) gave2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(5,6-dimethoxypyridin-2-yl)acetamide(9 mg, 0.024 mmol, 54.7% yield) ¹HNMR (400 MHz, CD₃OD) δ ppm 8.51 (s,1H), 7.58 (d, J=8.4 Hz, 1H), 7.51 (s, 1H), 7.22 (d, J=8.4 Hz, 1H), 4.23(s, 2H), 3.89 (s, 3H), 3.80 (s, 3H). ESI-MS m/z, 380.09 (M+H)⁺

2-Chloro-N-(4-methoxy-3-((2,2,2-trifluoroethoxy)phenyl)acetamide(Compound 88)

To the solution of 4-methoxy-3-((2,2,2-trifluoroethoxy)aniline (50 mg,0.226 mmol), TEA (0.079 mL, 0.565 mmol) in DCM (2 mL) was added2-chloroacetyl chloride (0.020 mL, 0.249 mmol) at room temperature.After 1 hr, concentration and purification on combiflash (4 g,EtOAc/Hexane) gave2-chloro-N-(4-methoxy-3-((2,2,2-trifluoroethoxy)phenyl)acetamide (49 mg,0.165 mmol, 72.8% yield). ¹HNMR (400 MHz, CDCl₃) δ ppm 8.13 (s, 1H),7.36 (s, 1H), 7.26 (s, 1H), 7.13 (d, J=8.8 Hz, 1H), 6.89 (d, J=8.8 Hz,1H), 4.39 (m, 2H), 4.18 (s, 2H), 3.87 (s, 3H) ESI-MS: m/z 297.92 (M+H)⁺

2-((6-Chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-methoxy-3-((2,2,2-trifluoroethoxy)phenyl)acetamide(Compound 89)

The reaction mixture of 6-chloro-1H-imidazo[4,5-c]pyridine-2-thiol (10mg, 0.054 mmol),2-chloro-N-(4-methoxy-3-((2,2,2-trifluoroethoxy)phenyl)acetamide (16.04mg, 0.054 mmol), sodium iodide (1 mg, 6.67 μmol) and triethylamine(0.023 mL, 0.162 mmol) in CH₃CN (1 mL) was heated to 65° C. Two newsports was purified by pre-TLC (MeOH/DCM) gave2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-methoxy-3-((2,2,2-trifluoroethoxy)phenyl)acetamide(9 mg, 0.020 mmol, 37.4% yield) ¹HNMR (400 MHz, CD₃OD) δ ppm 8.50 (s,1H), 7.50 (s, 1H), 7.37 (s, 1H), 7.16 (d, J=8.8 Hz, 1H), 6.97 (d, J=8.8Hz, 1H), 4.47 (m, 2H), 4.22 (s, 2H), 3.83 (s, 3H). ESI-MS m/z, 447.01(M+H)⁺

2-Chloro-N-(5,6-dimethoxypyridin-3-yl)acetamide (Compound 90)

To the solution of 5,6-dimethoxypyridin-3-amine (50 mg, 0.324 mmol), TEA(0.113 mL, 0.811 mmol) in DCM (3 mL) was added 2-chloroacetyl chloride(0.028 mL, 0.357 mmol) at room temperature. After 1 hr, concentrationand purification on combiflash (4 g, EtOAc/Hexane) gave2-chloro-N-(5,6-dimethoxypyridin-3-yl)acetamide (56 mg, 0.243 mmol,74.9% yield). ¹HNMR (400 MHz, CDCl₃) δ ppm 8.16 (s, 1H), 7.71 (s, J=2Hz, 1H), 7.62 (d, J=2 Hz, 1H), 4.21 (s, 2H), 4.01 (s, 3H), 3.90 (s, 3H)ESI-MS: m/z 230.99 (M+H)⁺

2-((6-Chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(5,6-dimethoxypyridin-3-yl)acetamide(Compound 91)

The reaction mixture of 6-chloro-1H-imidazo[4,5-c]pyridine-2-thiol (8.05mg, 0.043 mmol), 2-chloro-N-(5,6-dimethoxypyridin-3-yl)acetamide (10 mg,0.043 mmol), sodium iodide (1 mg, 6.67 μmol) and triethylamine (0.018mL, 0.130 mmol) in CH₃CN (1 mL) was heated to 65° C. overnight. Two newsports was purified by pre-TLC (MeOH/DCM) gave2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(5,6-dimethoxypyridin-3-yl)acetamide(13 mg, 0.034 mmol, 79% yield) 1HNMR (400 MHz, CD₃OD) δ ppm 8.50 (s,1H), 7.82 (d, J=2 Hz, 1H), 7.58 (d, J=2 Hz, 1H), 7.50 (s, 1H), 4.25 (s,2H), 3.91 (s, 3H), 3.82 (s, 3H), ESI-MS m/z, 380.04 (M+H)⁺

2-((6-Cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-hydroxyphenyl)acetamide(Compound 92)

A mixture of2-mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (11.21 mg,0.059 mmol), 2-chloro-N-(4-fluoro-3-hydroxyphenyl)acetamide (10 mg,0.049 mmol) and sodium iodide (3.68 mg, 0.025 mmol) in MeCN was addedtriethylamine (10.27 μl, 0.074 mmol) and stirred at 65° C. for 1 h. MeCNwas removed and the crude was purified by combiflash SiO₂ chromatography(0-5% MeOH-DCM) to afford2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-hydroxyphenyl)acetamide(10 mg, 57%) as a white solid. ¹H-NMR (400 MHz, CDCl₃—CD₃OD) δ ppm 8.22(s, 1H), 6.82 (dd, 1H, J=2.4, 8.0 Hz), 6.49 (m, 2H), 3.76 (s, 2H), 2.29(s, 3H); ESI-MS: m/z 358.03 (M+H)⁺.

2-((6-Cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-fluoro-4-methoxyphenyl)acetamide(Compound 93)

A mixture of 2-chloro-N-(3-fluoro-4-methoxyphenyl)acetamide (13.73 mg,0.063 mmol),2-mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (10 mg,0.053 mmol), sodium iodide (3.94 mg, 0.026 mmol) in MeCN was addedtriethylamine (10.99 μl, 0.079 mmol) and heated to 65° C. for 1 hr. MeCNwas removed and the crude was purified by combiflash SiO₂ chromatography(0-5% MeOH-DCM) to give2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-fluoro-4-methoxyphenyl)acetamide(11 mg, 56%) as a white solid. ¹H-NMR (400 MHz, CDCl₃—CD₃OD) δ ppm 8.65(s, 1H), 7.49 (dd, 1H, J=2.8, 13.2 Hz), 7.18 (d, 1H, J=8.4 Hz), 7.01 (t,1H, J=9.2 Hz), 4.24 (s, 2H), 3.83 (s, 3H), 2.70 (s, 3H); ESI-MS: m/z372.02 (M+H)⁺.

6-Amino-2-methoxypyridin-3-ol (Compound 94)

To a cooled solution of 5,6-dimethoxypyridin-2-amine (70 mg, 0.454 mmol)in DCM, tribromoborane (1.362 ml, 1.362 mmol) (1M in DCM) was slowlyadded and the mixture was stirred at room temperature for 3 hr. Thereaction mixture was quenched with sat.Na₂CO₃ followed by the additionof water. The aqueous layer was extracted with DCM, and the combinedorganic layers were washed with water, brine, dried over Na₂SO₄ andconcentrated under reduced pressure. The crude was purified bycombiflash SiO₂ chromatography to give 6-amino-2-methoxypyridin-3-ol (24mg, 38%) as a brown solid. ¹H-NMR (400 MHz, CD₃OD) δ ppm 6.91 (d, 1H,J=8 Hz), 6.03 (d, 1H, J=8.4 Hz), 3.87 (s, 3H); ESI-MS m/z 141.17 (M+H)⁺.

2-Chloro-N-(5-hydroxy-6-methoxypyridin-2-yl)acetamide (Compound 95)

To a cooled mixture of 6-amino-2-methoxypyridin-3-ol (24 mg, 0.171 mmol)and potassium carbonate (28.4 mg, 0.206 mmol) in acetone-water,2-chloroacetyl chloride (0.015 ml, 0.188 mmol) was slowly added at 0° C.and stirred for 1 h. Reaction was diluted with EtOAc and washed withwater, brine, dried over Na₂SO₄ and evaporated. The crude,2-chloro-N-(5-hydroxy-6-methoxypyridin-2-yl)acetamide (33 mg, 89%) as abrown solid, was used in the next step without further purification.¹H-NMR (400 MHz, CD₃OD) δ ppm 7.50 (d, 1H, J=8.4 Hz), 7.07 (d, 1H, J=8.4Hz), 4.19 (s, 2H), 3.93 (s, 3H); ESI-MS: m/z 217.00 (M+H)⁺.

2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(5-hydroxy-6-methoxypyridin-2-yl)acetamide(Compound 96)

To a mixture of 2-chloro-N-(5-hydroxy-6-methoxypyridin-2-yl)acetamide(10 mg, 0.046 mmol),2-mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (10.54 mg,0.055 mmol) and sodium iodide (3.46 mg, 0.023 mmol) in MeCN,triethylamine (9.65 μl, 0.069 mmol) was added and the reaction wasstirred at 65° C. for 1 hr. Solvent was removed and the crude waspurified by combiflash SiO₂ chromatography (0-5% MeOH-DCM) to give2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(5-hydroxy-6-methoxypyridin-2-yl)acetamide(13 mg, 76%) as a white solid. ¹H-NMR (400 MHz, DMSO-d₆) δ ppm 13.69 (s,1H), 10.43 (s, 1H), 9.22 (s, 1H), 8.70 (s, 1H), 7.40 (d, II-1, J=8.4Hz), 7.05 (d, 1H, J=8.0 Hz), 4.34 (s, 2H), 3.84 (s, 3H), 2.62 (s, 3H);ESI-MS: m/z 371.07 (M+H)⁺.

Experimental Biology:

1. ENPP1 Inhibition Assay

Materials:

-   -   Assay Buffer: 1 mM CaCl2, 0.2 mM ZnCl2, 50 mM Tris, pH 9.0    -   Substrate: 8 mM Thymidine 5′-monophosphate p-nitrophenol ester        sodium salt (Sigma Cat # T4510)    -   Enzyme: 5 ng/μL Recombinant Human ENPP-1 Protein (R&D Cat        #6136-EN-010) DMSO    -   96-well clear assay plates

Methods:

An eight point serial dilution of drugs was prepared in 10× in assaybuffer with the final assay concentrations starting at 10 μM, 3 μM, 1μM, 0.3 μM . . . 0 μM. A dilution of DMSO was included as a control. Theassay plate was set up as follows with each well in duplicate: 81 μLassay buffer+10 μL ENPP1 inhibitor or DMSO+5 μL Substrate+4 μL Enzyme.Both the enzyme and substrate were added to opposite sides of the wellto ensure that there was no interaction until all wells had bothcomponents. The plate was then centrifuged gently for 10 seconds,followed by an incubation at 37° C. for 45 minutes. The reaction wasquantified by measuring absorbance at 405 nm using the Envision.

IC₅₀ Calculation:

IC₅₀ values are determined using GraphPad Prism 5 software. The datawere entered as an X-Y plot into the software as percent inhibition foreach concentration of the drug. The concentration values of the drugwere log transformed and the nonlinear regression was carried out usingthe “sigmoidal dose-response (variable slope)” option within theGraphPad software to model the data and calculate IC₅₀ values. The IC₅₀values reported are the concentration of drug at which 50% inhibitionwas reached.

The results of this experiment are shown in FIG. 1. FIG. 1 demonstratesthat compounds 21, 68 and 78 were effective in inhibiting the activityof ENPP1.

2. ENPP2 Inhibition Assay

Materials:

Assay Buffer: 10 mM CaCl₂, 5 mM MgCl₂, 50 mM Tris, 0.02% Brij-35, pH 8.5

Substrate: 2 mM Bis(p-Nitrophenyl) Phosphate Sodium Salt (Sigma Cat #N3002)

Enzyme: 0.2 ng/μL Recombinant Human ENPP-2/Autotaxin Protein (R&D Cat#5255-EN)

PF-3080

DMSO

96-well clear assay plates

Methods:

An eight point serial dilution of drugs was prepared in 10× in assaybuffer with the final assay concentrations starting at 10 μM, 3 μM, 1μM, 0.3 μM . . . 0 μM. A dilution of DMSO was included as a control. Theassay plate was set up as follows with each well in duplicate: 15 μLassay buffer+10 μL, ENPP1 inhibitor or DMSO+50 μL Substrate+25 μLEnzyme. Both the enzyme and substrate were added to opposite sides ofthe well to ensure that there was no interaction until all wells hadboth components. The plate was then centrifuged gently for 10 seconds,followed by an incubation at 37° C. for 45 minutes. The reaction wasquantified by measuring absorbance at 405 nm using the Envision.

IC₅₀ Calculation:

IC₅₀ values are determined using GraphPad Prism 5 software. The datawere entered as an X-Y plot into the software as percent inhibition foreach concentration of the drug. The concentration values of the drugwere log transformed and the nonlinear regression was carried out usingthe “sigmoidal dose-response (variable slope)” option within theGraphPad software to model the data and calculate IC₅₀ values. The IC₅₀values reported are the concentration of drug at which 50% inhibitionwas reached.

The results of this experiment are shown in FIGS. 2A and 2B. FIGS. 2Aand 2B demonstrate that compounds 21 and 78 and their respective sodiumsalts were ineffective in inhibiting the activity of ENPP2. Theseresults indicate that compounds 21 and 78 were selective at inhibitingENPP1 but not ENPP2.

3. ENPP1 Thermal Shift Assay

Materials:

Recombinant Human ENPP-1 Protein (R&D Cat #6136-EN-010)

Assay Buffer (1 mM CaCl2, 0.2 mM ZnCl2, 50 mM Tris, pH 9.0)

5000× SYPRO Orange (ThermoFisher cat # S6651)

384-well PCR Plates

Methods:

Each drug was prepared as a 10× solution in the assay buffer and SYPROOrange was diluted to 10× concentration in water. Wells were set up induplicate in a 384-well PCR plate as follows: 14 μL assay buffer, 2 μLENPP1 Inhibitor or DMSO, 2 μL (0.5 μg) ENPP1 protein. Each well wasmixed and incubated on ice for 5 minutes. Post incubation, 2 μL of SYPROOrange was mixed into each well and followed by a gentle centrifugation.The protein melt reaction was run using ViiA7 software with temperaturesbeginning at 25° C. and increasing by 0.05° C./s to the maximumtemperature of 99° C.

The results of this experiment are shown in FIG. 3. FIG. 3 demonstratesthat compound 21 was effective in increasing the stability ofrecombinant ENPP1 protein. The Tm (midpoint of the protein unfoldingtransition) of ENPP1 was 64.2° C. and this was increased to 67.84° C. inthe presence of compound 21. These results suggest that compound 21binds directly with ENPP1 and modulates its activity.

4. ENPP1 Mineralization Assay

Materials:

Saos-2 Cells (ATCC CAT # HTB-85)

Culture Medium

24-well Tissue Culture Plate

Ascorbic Acid, 5 mg/mL (100×)

β-Glycerophosphate, 1M (100×)

Phosphate Buffered Saline

Alizarin Red, 40 mM pH 4.2

70% Ethanol

Dye Extraction Reagent, 10% Methanol+10% Acetic Acid

Methods:

Saos-2 cells were plated at a density of 1.5×106 cells/well and allowedto grow to 100% confluency over the following 1-2 days. Once completelyconfluent, the media was replaced with fresh media supplemented with 1×ascorbic acid, 1× β-glycerophosphate, and varying concentrations ofENPP1 inhibitor (if applicable). The addition of the mineralizationcomponents was considered Day 1, On Day 3, the media was gentlyaspirated with no wash followed by the addition of fresh media,mineralization components, and ENPP1 inhibitor (if applicable). On day5, the media was gently aspirated and the cells were washed twice withPBS. The cells were then fixed with ice cold 70% ethanol for 1 hour at4° C. Cells were washed once with PBS and stained with Alizarin Red for30 minutes at room temperature with gentle rotation. Stained cells werewashed three times with water for 10 minutes at room temperature withgentle rotation. After imaging cells, dye extraction reagent was addedto each well and incubated for 30 minutes at room temperature. Alizarinred was quantified by measuring absorbance at a wavelength of 405 nmusing the Envision.

The results of this experiment are shown in FIG. 4. FIG. 4 demonstratesthat compounds 21 and 68 were effective in inhibiting ENPP1 ability toproduce mineralized nodules. These results suggest that inhibition ofENPP1 likely altered the pyrophosphate/phosphate ratio, which waspreviously reported to regulate matrix mineralization of cells.

The ability of representative disclosed compounds to modulate variousbiochemical and cellular activities was determined using the ENPP1 andENPP2 inhibition assay, ENPP1 protein stability assay and ENPP1 mineralassay described above.

The IC₅₀ (μM) for inhibition of either ENPP1 activity are shown inTables 1 and 2.

If an IC₅₀ or another assay result is indicated as “ND”, it was notdetermined in the indicated assay.

TABLE 1 3H-Imidazo [4,5-c] Pyridine Series 1 of ENPP1 Inhibitors ENPP1ENPP1 Mineral Compound inhibition assay (50% Number Structure MW. (IC₅₀)(μM) inhibition at 50 μM) 3

C₁₆H₁₅ClN₄O₃S 378.83 0.58 NT 7

C₁₅H₁₁ClN₄O₃S 362.79 >50.0 NT 14

C₁₇H₁₇ClN₄O₃S 392.86 3.522 NT 21

C₁₅H₁₃ClN₄O₃S 364.80 0.086 + 22

C₁₅H₁₃ClN₄O₃S 364.80 1.161 + 25

C₁₄H₁₁ClN₄O₂S 334.78 7.932 NT 40

C₁₅H₁₁ClF₂N₄O₂S 384.79 33.61 NT 27

C₁₅H₁₂ClFN₄O₂S 366.80 0.400 NT 29

C₁₆H₁₃ClF₂N₄O₃S 414.81 1.162 NT 31

C₁₆H₁₃F₃N₄O₃S 398.36 0.181 + 33

C₁₄H₁₁ClN₄O₂S 334.78 7.921 NT 35

C₁₆H₁₂ClF₃N₄O₃S 432.80 40.69 NT 37

C₁₄H₁₀ClFN₄O₂S 352.77 0.170 + 38

C₁₆H₁₂F₄N₄O₂S 400.35 0.487 + 42

C₁₇H₁₅F₃N₄O₃S 412.39 11.35 NT 44

C₁₆H₁₁F₅N₄O₂S 418.34 10.2 NT 45

C₁₅H₁₀F₄N₄O₂S 386.32 0.651 + 61

C₁₆H₁₀F₆N₄O₃S 452.33 22.61 NT 49

C₁₆H₁₅ClN₄O₃S 378.83 0.667 NT 50

C₁₆H₁₄ClFN₄O₂S 380.82 5.627 NT 51

C₁₅H₁₂ClFN₄O₂S 366.80 1.269 NT 52

C₁₇H₁₇ClN₄O₃S 392.86 1.138 NT 62

C₁₅H₁₀ClN₅OS₂ 375.85 10.86 NT 67

C₁₆H₁₃N₅O₃S 355.3720 0.179 NT 54

C₁₇H₁₄ClN₅O₃S₂ 435.90 5.728 NT 68

C₁₇H₁₅N₅O₃S 369.40 0.114 + 69

C₁₅H₁₀FN₅O₂S 343.34 0.216 NT 70

C₁₅H₁₂ClFN₄O₄S 398.79 30.18 NT 77

C₁₈H₁₇N₅O₃S 383.42 0.265 NT 78

C₁₇H₁₅N₅O₃S 369.40 0.079 + 72

C₁₄H₁₂ClN₅O₂S 349.79 3.295 NT 82

C₁₅H₁₄ClN₅O₂S 363.82 6.02 NT 83

C₁₅H₁₂ClN₅O₄S 393.81 4.55 NT 79

C₁₆H₁₂FN₅O₂S 357.36 6.521 NT 85

C₁₅H₁₄ClN₅O₃S 379.82 0.266 NT 93

C₁₇H₁₄FN₅O₂S 371.39 0.224 NT 87

C₁₅H₁₄ClN₅O₃S 379.82 1.977 NT 89

C₁₇H₁₄ClF₃N₄O₃S 446.83 11.32 NT 96

C₁₆H₁₄N₆O₃S 370.39 2.058 NT 91

C₁₅H₁₄ClN₅O₃S 379.8 NT NT

TABLE 2 3H-Imidazo [4,5-c] Pyridine Series 1 of ENPP1 Inhibitors 9

C₁₇H₁₈N₄O₃S 358.42 4.459 NT 10

C₁₆H₁₄N₄O₃S 342.37 50.0 NT 16

C₁₆H₁₆N₄O₃S 344.4 2.294 NT 18

C₁₆H₁₆N₄O₃S 344.4 8.371 NT NA: Not Active; NT: Not Tested; +: active at50 μM concentration

Prophetic Pharmaceutical Composition Examples

“Active ingredient” as used throughout these examples relates to one ormore of the compounds of the invention, or a pharmaceutically acceptablesalt, solvate, polymorph, hydrate and the stereochemically isomeric formthereof. The following examples of the formulation of the compounds ofthe present invention in tablets, suspension, injectables and ointmentsare prophetic.

Typical examples of recipes for the formulation of the invention are asgiven below. Various other dosage forms can be applied herein such as afilled gelatin capsule, liquid emulsion/suspension, ointments,suppositories or chewable tablet form employing the disclosed compoundsin desired dosage amounts in accordance with the present invention.Various conventional techniques for preparing suitable dosage forms canbe used to prepare the prophetic pharmaceutical compositions, such asthose disclosed herein and in standard reference texts, for example theBritish and US Pharmacopoeias, Remington's Pharmaceutical Sciences (MackPublishing Co.) and Martindale The Extra Pharmacopoeia (London ThePharmaceutical Press). The disclosure of this reference is herebyincorporated herein by reference.

a. Pharmaceutical Composition for Oral Administration

A tablet can be prepared as follows:

Component Amount Active ingredient 10 to 500 mg Lactose 100 mgCrystalline cellulose 60 mg Magnesium stearate 5 Starch (e.g. potatostarch) Amount necessary to yield total weight indicated below Total(per capsule) 1000 mg

Alternatively, about 100 mg of a disclosed compound, 50 mg of lactose(monohydrate), 50 mg of maize starch (native), 10 mg ofpolyvinylpyrrolidone (PVP 25) (e.g. from BASF, Ludwigshafen, Germany)and 2 mg of magnesium stearate are used per tablet. The mixture ofactive component, lactose and starch is granulated with a 5% solution(m/m) of the PVP in water. After drying, the granules are mixed withmagnesium stearate for 5 min. This mixture is moulded using a customarytablet press (e.g. tablet format: diameter 8 mm, curvature radius 12mm). The moulding force applied is typically about 15 kN.

Alternatively, a disclosed compound can be administered in a suspensionformulated for oral use. For example, about 100-5000 mg of the desireddisclosed compound, 1000 mg of ethanol (96%), 400 mg of xanthan gum, and99 g of water are combined with stirring. A single dose of about 10-500mg of the desired disclosed compound according can be provided by 10 mlof oral suspension.

In these Examples, active ingredient can be replaced with the sameamount of any of the compounds according to the present invention, inparticular by the same amount of any of the exemplified compounds. Insome circumstances it may be desirable to use a capsule, e.g. a filledgelatin capsule, instead of a tablet form. The choice of tablet orcapsule will depend, in part, upon physicochemical characteristics ofthe particular disclosed compound used.

Examples of alternative useful carriers for making oral preparations arelactose, sucrose, starch, talc, magnesium stearate, crystallinecellulose, methyl cellulose, hydroxypropyl cellulose,hydroxypropylmethyl cellulose, carboxymethyl cellulose, glycerin, sodiumalginate, gum arabic, etc. These alternative carriers can be substitutedfor those given above as required for desired dissolution, absorption,and manufacturing characteristics.

The amount of a disclosed compound per tablet for use in apharmaceutical composition for human use is determined from bothtoxicological and pharmacokinetic data obtained in suitable animalmodels, e.g. rat and at least one non-rodent species, and adjusted basedupon human clinical trial data. For example, it could be appropriatethat a disclosed compound is present at a level of about 10 to 1000 mgper tablet dosage unit.

b. Pharmaceutical Composition for Injectable Use

A parenteral composition can be prepared as follows:

Component Amount Active ingredient 10 to 500 mg Sodium carbonate 560 mg*Sodium hydroxide 80 mg* Distilled, sterile water Quantity sufficient toprepare total volumen indicated below. Total (per capsule) 10 ml perampule *Amount adjusted as required to maintain physiological pH in thecontext of the amount of active ingredient, and form of activeingredient, e.g. a particular salt form of the active ingredient.

Alternatively, a pharmaceutical composition for intravenous injectioncan be used, with composition comprising about 100-5000 mg of adisclosed compound, 15 g polyethylenglycol 400 and 250 g water in salinewith optionally up to about 15% Cremophor EL, and optionally up to 15%ethyl alcohol, and optionally up to 2 equivalents of a pharmaceuticallysuitable acid such as citric acid or hydrochloric acid are used. Thepreparation of such an injectable composition can be accomplished asfollows: The disclosed compound and the polyethylenglycol 400 aredissolved in the water with stirring. The solution is sterile filtered(pore size 0.22 μm) and filled into heat sterilized infusion bottlesunder aseptic conditions. The infusion bottles are sealed with rubberseals.

In a further example, a pharmaceutical composition for intravenousinjection can be used, with composition comprising about 10-500 mg of adisclosed compound, standard saline solution, optionally with up to 15%by weight of Cremophor EL, and optionally up to 15% by weight of ethylalcohol, and optionally up to 2 equivalents of a pharmaceuticallysuitable acid such as citric acid or hydrochloric acid. Preparation canbe accomplished as follows: a desired disclosed compound is dissolved inthe saline solution with stirring. Optionally Cremophor EL, ethylalcohol or acid are added. The solution is sterile filtered (pore size0.22 μm) and filled into heat sterilized infusion bottles under asepticconditions. The infusion bottles are sealed with rubber seals.

In this Example, active ingredient can be replaced with the same amountof any of the compounds according to the present invention, inparticular by the same amount of any of the exemplified compounds.

The amount of a disclosed compound per ampule for use in apharmaceutical composition for human use is determined from bothtoxicological and pharmacokinetic data obtained in suitable animalmodels, e.g. rat and at least one non-rodent species, and adjusted basedupon human clinical trial data. For example, it could be appropriatethat a disclosed compound is present at a level of about 10 to 1000 mgper tablet dosage unit.

Carriers suitable for parenteral preparations are, for example, water,physiological saline solution, etc. which can be used withtris(hydroxymethyl)aminomethane, sodium carbonate, sodium hydroxide orthe like serving as a solubilizer or pH adjusting agent. The parenteralpreparations contain preferably 50 to 1000 mg of a disclosed compoundper dosage unit.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

What is claimed is:
 1. A compound of structure:

or a pharmaceutically acceptable salt thereof.
 2. A pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof claim 1 and a pharmaceutically acceptable carrier.
 3. A method forthe treatment of a disorder of uncontrolled cellular proliferation in amammal, the method comprising the step of administering to the mammal aneffective amount of a compound of claim
 1. 4. A method for treatingcancer in a mammal, the method comprising the step of administering tothe mammal an effective amount of a compound of claim 1.